Genes and proteins of Brachyspira hyodysenteriae and uses thereof

ABSTRACT

Novel polynucleotide and amino acids of  Brachyspira hyodysenteriae  are described. These sequences are useful for diagnosis of  B. hyodysenteriae  disease in animals and as a therapeutic treatment or prophylactic treatment of  B. hyodysenteriae  disease in animals. These sequences may also be useful for diagnostic and therapeutic and/or prophylactic treatment of diseases in animals caused by other  Brachyspira  species, including  B. intermedia, B. suantatina, B. alvinipulli, B. aalborgi, B. innocens, B. murdochii , and  B. pilosicoli.

FIELD OF INVENTION

This invention relates to novel genes in Brachyspira hyodysenteriae andthe proteins encoded therein. This invention further relates to use ofthese novel genes and proteins for diagnosis of B. hyodysenteriaedisease, vaccines against B. hyodysenteriae and for screening forcompounds that kill B. hyodysenteriae or block the pathogenic effects ofB. hyodysenteriae. These sequences may also be useful for diagnostic andtherapeutic and/or prophylactic treatment of diseases in animals causedby other Brachyspira species, including B. suanatina, B. intermedia, B.alvinipulli, B. aalborgi, B. innocens, B. murdochii, and B. pilosicoli.

BACKGROUND OF INVENTION

Swine dysentery is a significant endemic disease of pigs in Australiaand worldwide. Swine dysentery is a contagious mucohaemorrhagicdiarrhoeal disease, characterised by extensive inflammation and necrosisof the epithelial surface of the large intestine. Economic losses due toswine dysentery result mainly from growth retardation, costs ofmedication and mortality. The causative agent of swine dysentery wasfirst identified as an anaerobic spirochaete (Treponema hyodysenteriae)in 1971, and was recently reassigned to the genus Brachyspira as B.hyodysenteriae. Where swine dysentery is established in a piggery, thedisease spectrum can vary from being mild, transient or unapparent, tobeing severe and even fatal. Medication strategies on individualpiggeries may mask clinical signs and on some piggeries the disease maygo unnoticed, or may only be suspected. Whether or not obvious diseaseoccurs, B. hyodysenteriae may persist in infected pigs, or in otherreservoir hosts such as rodents, or in the environment. All thesesources pose potential for transmission of the disease to uninfectedherds. Commercial poultry may also be colonized by B. hyodysenteriae,although it is not clear how commonly this occurs under fieldconditions.

Colonisation by B. hyodysenteriae elicits a strong immunologicalresponse against the spirochaete, hence indirect evidence of exposure tothe spirochaete can be obtained by measuring circulating antibody titresin the blood of infected animals. These antibody titres have beenreported to be maintained at low levels, even in animals that haverecovered from swine dysentery. Serological tests for detection ofantibodies therefore have considerable potential for detectingsubclinical infections and recovered carrier pigs that have undetectablenumbers of spirochaetes in their large intestines. These tests would beparticularly valuable in an easy to use kit form, such as anenzyme-linked immunosorbent assay. A variety of techniques have beendeveloped to demonstrate the presence of circulating antibodies againstB. hyodysenteriae, including indirect fluorescent antibody tests,haemagglutination tests, microtitration agglutination tests, complementfixation tests, and ELISA using either lipopolysaccharide or wholesonicated spirochaetes as antigen. All these tests have suffered fromproblems of specificity, as related non-pathogenic intestinalspirochaetes can induce cross-reactive antibodies. These tests areuseful for detecting herds where there is obvious disease and highcirculating antibody titres, but they are problematic for identifyingsub-clinically infected herds and individual infected pigs.Consequently, to date, no completely sensitive and specific assays areavailable for the detection of antibodies against B. hyodysenteriae. Thelack of suitable diagnostic tests has hampered control of swinedysentery.

A number of methods are employed to control swine dysentery, varyingfrom the prophylactic use of antimicrobial agents, to completedestocking of infected herds and prevention of re-entry of infectedcarrier pigs. All these options are expensive and, if they are to befully effective, they require the use of sophisticated diagnostic teststo monitor progress. Currently, detection of swine dysentery in herdswith sub-clinical infections, and individual healthy carrier animals,remains a major problem and is hampering implementation of effectivecontrol measures. A definitive diagnosis of swine dysenterytraditionally has required the isolation and identification of B.hyodysenteriae from the faeces or mucosa of diseased pigs. Majorproblems involved include the slow growth and fastidious nutritionalrequirements of these anaerobic bacteria and confusion due to thepresence of morphologically similar spirochaetes in the normal flora ofthe pig intestine. A significant improvement in the diagnosis ofindividual affected pigs was achieved with the development of polymerasechain reaction (PCR) assays for the detection of spirochaetes fromfaeces. Unfortunately in practical applications the limit of detectionof PCRs rendered it unable to detect carrier animals with subclinicalinfections. As a consequence of these diagnostic problems, there is aclear need to develop a simple and effective diagnostic tool capable ofdetecting B. hyodysenteriae infection at the herd and individual piglevel.

A strong immunological response is induced against the spirochaetefollowing colonization with B. hyodysenteriae, and pigs recovered fromswine dysentery are protected from re-infection. Despite this, attemptsto develop vaccines to control swine dysentery have met with verylimited success, either because they have provided inadequate protectionon a herd basis, or they have been too costly and difficult to produceto make them commercially viable. Bacterin vaccines provide some levelof protection, but they tend to be lipopolysaccharideserogroup-specific, which then requires the use of multivalentbacterins. Furthermore they are difficult and costly to produce on alarge scale because of the fastidious anaerobic growth requirements ofthe spirochaete.

Several attempts have been made to develop attenuated live vaccines forswine dysentery. This approach has the disadvantage that attenuatedstrains show reduced colonisation, and hence cause reduced immunestimulation. There also is reluctance on the part of producers andveterinarians to use live vaccines for swine dysentery because of thepossibility of reversion to virulence, especially as very little isknown about genetic regulation and organization in B. hyodysenteriae.

The use of recombinant subunit vaccines is an attractive alternative,since the products would be well-defined (essential for registrationpurposes), and relatively easy to produce on a large scale. To date thefirst reported use of a recombinant protein from B. hyodysenteriae as avaccine candidate (a 38-kilodalton flagellar protein) failed to preventcolonisation in pigs. This failure is likely to relate specifically tothe particular recombinant protein used, as well as to other moredown-stream issues of delivery systems and routes, dose rates, choice ofadjuvants etc. (Gabe, J D, Chang, R J, Slomiany, R, Andrews, W H andMcCaman, M T (1995) Isolation of extracytoplasmic proteins fromSerpulina hyodysenteriae B204 and molecular cloning of the flaB1 geneencoding a 38-kilodalton flagellar protein.

Infection and Immunity 63:142-148). The first reported partiallyprotective recombinant B. hyodysenteriae protein used for vaccinationwas a 29.7 kDa outer membrane lipoprotein (Bhlp29.7, also referred to asBmpB and BlpA) which had homology with the methionine-bindinglipoproteins of various pathogenic bacteria. The use of the his-taggedrecombinant Bhlp29.7 protein for vaccination of pigs, followed byexperimental challenge with B. hyodysenteriae, resulted in 17-40% ofvaccinated pigs developing disease compared to 50-70% of theunvaccinated control pigs developing disease. Since the incidence ofdisease for the Bhlp29.7 vaccinated pigs was significantly (P=0.047)less than for the control pigs, Bhlp29.7 appeared to have potential as aswine dysentery vaccine component (La, T, Phillips, N D, Reichel, M Pand Hampson, D J (2004). Protection of pigs from swine dysentery byvaccination with recombinant BmpB, a 29.7 kDa outer-membrane lipoproteinof Brachyspira hyodysenteriae. Veterinary Microbiology 102:97-109). Anumber of other attempts have been made to identify outer envelopproteins from B. hyodysenteriae that could be used as recombinantvaccine components, but again no successful vaccine has yet been made. Amuch more global approach is needed to the identification of potentiallyuseful immunogenic recombinant proteins from B. hyodysenteriae isneeded.

To date, only one study using DNA for vaccination has been reported. Inthis study, the B. hyodysenteriae ftnA gene, encoding a putativeferritin, was cloned into an E. coli plasmid and the plasmid DNA used tocoat gold beads for ballistic vaccination. A murine model for swinedysentery was used to determine the protective nature of vaccinationwith DNA and/or recombinant protein. Vaccination with recombinantprotein induced a good systemic response against ferritin howevervaccination with DNA induced only a detectable systemic response.Vaccination with DNA followed a boost with recombinant protein induced asystemic immune response to ferritin only after boosting with protein.However, none of the vaccination regimes tested was able to provide themice with protection against B. hyodysenteriae colonisation and theassociated lesions. Interestingly, vaccination of the mice with DNAalone resulted in significant exacerbation of disease (Davis, A. J.,Smith, S. C. and Moore, R. J. (2005). The Brachyspira hyodysenteriaeftnA gene: DNA vaccination and real-time PCR quantification of bacteriain a mouse model of disease. Current Microbiology 50: 285-291).

BRIEF SUMMARY OF INVENTION

It is an object of this invention to have novel genes from B.hyodysenteriae and the proteins encoded by those genes. It is a furtherobject of this invention that the novel genes and the proteins encodedby those genes can be used for therapeutic and diagnostic purposes. Onecan use the genes and/or the proteins in a vaccine against B.hyodysenteriae and to diagnose B. hyodysenteriae infections.

It is an object of this invention to have novel B. hyodysenteriae geneshaving the nucleotide sequence contained in SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, and 65. It is also an object of thisinvention to have nucleotide sequences that are identical to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, and 65 where thepercentage identity can be at least 95%, 90%, 85%, 80%, 75% and 70% (andevery integer from 100 to 70). This invention also includes a DNAvaccine or DNA immunogenic composition containing the nucleotidesequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, and 65 and sequences that are at least 95%, 90%, 85%, 80%, 75% and70% identical (and every integer from 100 to 70) to these sequences.This invention further includes a diagnostic assay containing DNA havingthe nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, and 65 and sequences that are at least 95%, 90%,85%, 80%, 75% and 70% identical (and every integer from 100 to 70) tothese sequences.

It is also an object of this invention to have plasmids containing DNAhaving the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, and 65; prokaryotic and/or eukaryotic expression vectorscontaining DNA having the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, and 65; and a cell containing the plasmidswhich contain DNA having the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, and 65.

It is an object of this invention to have novel B. hyodysenteriaeproteins having the amino acid sequence contained in SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, and 66. It is anotherobject of this invention to have proteins that are at least 95%, 90%,85%, 80%, 75% and 70% homologous (and every integer from 100 to 70) tothe sequences contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, and 66. It is also an object of this invention for avaccine or immunogenic composition to contain the proteins having theamino acid sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, and 66, or amino acid sequences that are atleast 95%, 90%, 85%, 80%, 75% and 70% homologous (and every integer from100 to 70) to the sequences contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, and 66. It is a further aspect of thisinvention to have a diagnostic kit containing one or more proteinshaving a sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, and 66 or that are at least 95%, 90%, 85%, 80%,75% and 70% homologous to the sequences contained in SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, and 66.

It is another aspect of this invention to have nucleotide sequenceswhich encode the proteins having the amino acid sequence contained inSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, and 66,and encode the amino acid sequences that are at least 95%, 90%, 85%,80%, 75% and 70% homologous (and every integer from 100 to 70) to thesequences contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, and 66. The invention also covers plasmids, eukaryoticand prokaryotic expression vectors, and DNA vaccines which contain DNAhaving a sequence which encodes a protein having the amino acid sequencecontained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, and 66, and encode amino acid sequences that are at least 95%, 90%,85%, 80%, 75% and 70% homologous (and every integer from 100 to 70) tothe sequences contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, and 66. Cells which contain these plasmids andexpression vectors are included in this invention.

This invention includes monoclonal antibodies that bind to proteinshaving an amino acid sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, and 66 or bind to proteins that areat least 95%, 90%, 85%, 80%, 75% and 70% homologous (and every integerfrom 100 to 70) to the sequences contained in SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, and 66. Diagnostic kitscontaining the monoclonal antibodies that bind to proteins having anamino acid sequence contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, and 66 or bind to proteins that are at least95%, 90%, 85%, 80%, 75% and 70% homologous (and every integer from 100to 70) to the sequences contained in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, and 66 are included in this invention. Thesediagnostic kits can detect the presence of B. hyodysenteriae in ananimal. The animal is preferably any mammal and bird; more preferably,chicken, goose, duck, turkey, parakeet, dog, cat, hamster, gerbil,rabbit, ferret, horse, cow, sheep, pig, monkey, and human.

The invention also contemplates the method of preventing or treating aninfection of B. hyodysenteriae in an animal by administering to ananimal a DNA vaccine containing one or more nucleotide sequences listedin SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, and65 or sequences that are at least 95%, 90%, 85%, 80%, 75% and 70%identical (and every integer from 100 to 70) to these sequences. Thisinvention also covers a method of preventing or treating an infection ofB. hyodysenteriae in an animal by administering to an animal a vaccinecontaining one or more proteins having the amino acid sequencecontaining in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, and 66 or sequences that are at least 95%, 90%, 85%, 80%, 75%and 70% homologous (and every integer from 100 to 70) to thesesequences. The animal is preferably any mammal and bird; morepreferably, chicken, goose, duck, turkey, parakeet, dog, cat, hamster,gerbil, rabbit, ferret, horse, cow, sheep, pig, monkey, and human.

The invention also contemplates the method of generating an immuneresponse in an animal by administering to an animal an immunogeniccomposition containing one or more nucleotide sequences listed in SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, and 65 orsequences that are at least 95%, 90%, 85%, 80%, 75% and 70% identical(and every integer from 100 to 70) to these sequences. This inventionalso covers a method of generating an immune response in an animal byadministering to an animal an immunogenic composition containing one ormore proteins having the amino acid sequence containing in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, and 66 or sequencesthat are at least 95%, 90%, 85%, 80%, 75% and 70% homologous (and everyinteger from 100 to 70) to these sequences. The animal is preferably anymammal and bird; more preferably, chicken, goose, duck, turkey,parakeet, dog, cat, hamster, gerbil, rabbit, ferret, horse, cow, sheep,pig, monkey, and human.

DETAILED SUMMARY OF INVENTION

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing.

An animal can be any mammal or bird. Examples of mammals include dog,cat, hamster, gerbil, rabbit, ferret, horse, cow, sheep, pig, monkey,and human. Examples of birds include chicken, goose, duck, turkey, andparakeet.

The term “conserved residue” refers to an amino acid that is a member ofa group of amino acids having certain common properties. The term“conservative amino acid substitution” refers to the substitution(conceptually or otherwise) of an amino acid from one such group with adifferent amino acid from the same group. A functional way to definecommon properties between individual amino acids is to analyze thenormalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz, G. E. and R. H. Schinner.,Principles of Protein Structure, Springer-Verlag). According to suchanalyses, groups of amino acids may be defined where amino acids withina group exchange preferentially with each other, and therefore resembleeach other most in their impact on the overall protein structure(Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure,Springer-Verlag). Examples of amino acid groups defined in this mannerinclude: (i) a positively-charged group containing Lys, Arg and His,(ii) a negatively-charged group containing Glu and Asp, (iii) anaromatic group containing Phe, Tyr and Trp, (iv) a nitrogen ring groupcontaining His and Trp, (v) a large aliphatic nonpolar group containingVal, Leu and De, (vi) a slightly-polar group containing Met and Cys,(vii) a small-residue group containing Ser, Thr, Asp, Asn, Gly, Ala,Glu, Gln and Pro, (viii) an aliphatic group containing Val, Leu, De, Metand Cys, and (ix) a small, hydroxyl group containing Ser and Thr.

A “fusion protein” or “fusion polypeptide” refers to a chimeric proteinas that term is known in the art and may be constructed using methodsknown in the art. In many examples of fusion proteins, there are twodifferent polypeptide sequences, and in certain cases, there may bemore. The polynucleotide sequences encoding the fusion protein may beoperably linked in frame so that the fusion protein may be translatedcorrectly. A fusion protein may include polypeptide sequences from thesame species or from different species. In various embodiments, thefusion polypeptide may contain one or more amino acid sequences linkedto a first polypeptide. In the case where more than one amino acidsequence is fused to a first polypeptide, the fusion sequences may bemultiple copies of the same sequence, or alternatively, may be differentamino acid sequences. The fusion polypeptides may be fused to theN-terminus, the C-terminus, or the N- and C-terminus of the firstpolypeptide. Exemplary fusion proteins include polypeptides containing aglutathione S-transferase tag (GST-tag), histidine tag (His-tag), animmunoglobulin domain or an immunoglobulin binding domain.

The term “isolated polypeptide” refers to a polypeptide, in certainembodiments prepared from recombinant DNA or RNA, or of synthetic originor natural origin, or some combination thereof, which (1) is notassociated with proteins that it is normally found with in nature, (2)is separated from the cell in which it normally occurs, (3) is free ofother proteins from the same cellular source, (4) is expressed by a cellfrom a different species, or (5) does not occur in nature. It ispossible for an isolated polypeptide exist but not qualify as a purifiedpolypeptide.

The term “isolated nucleic acid” and “isolated polynucleotide” refers toa polynucleotide whether genomic DNA, cDNA, mRNA, tRNA, rRNA, iRNA, or apolynucleotide obtained from a cellular organelle (such as mitochondriaand chloroplast), or whether from synthetic origin, which (1) is notassociated with the cell in which the “isolated nucleic acid” is foundin nature, or (2) is operably linked to a polynucleotide to which it isnot linked in nature. It is possible for an isolated polynucleotideexist but not qualify as a purified polynucleotide.

The term “nucleic acid” and “polynucleotide” refers to a polymeric formof nucleotides, either ribonucleotides or deoxyribonucleotides or amodified form of either type of nucleotide. The terms should also beunderstood to include, as equivalents, analogs of either RNA or DNA madefrom nucleotide analogs, and, as applicable to the embodiment beingdescribed, single-stranded (such as sense or antisense) anddouble-stranded polynucleotides.

The term “nucleic acid of the invention” and “polynucleotide of theinvention” refers to a nucleic acid encoding a polypeptide of theinvention. A polynucleotide of the invention may comprise all, or aportion of, a subject nucleic acid sequence; a nucleotide sequence atleast 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to asubject nucleic acid sequence (and every integer between 60 and 100); anucleotide sequence that hybridizes under stringent conditions to asubject nucleic acid sequence; nucleotide sequences encodingpolypeptides that are functionally equivalent to polypeptides of theinvention; nucleotide sequences encoding polypeptides at least about60%, 70%, 80%, 85%, 90%, 95%, 98%, 99% homologous or identical with asubject amino acid sequence (and every integer between 60 and 100);nucleotide sequences encoding polypeptides having an activity of apolypeptide of the invention and having at least about 60%, 70%, 80%,85%, 90%, 95%, 98%, 99% or more homology or identity with a subjectamino acid sequence (and every integer between 60 and 100); nucleotidesequences that differ by 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75or more nucleotide substitutions, additions or deletions, such asallelic variants, of a subject nucleic acid sequence; nucleic acidsderived from and evolutionarily related to a subject nucleic acidsequence; and complements of, and nucleotide sequences resulting fromthe degeneracy of the genetic code, for all of the foregoing and othernucleic acids of the invention. Nucleic acids of the invention alsoinclude homologs, e.g., orthologs and paralogs, of a subject nucleicacid sequence and also variants of a subject nucleic acid sequence whichhave been codon optimized for expression in a particular organism (e.g.,host cell).

The term “operably linked”, when describing the relationship between twonucleic acid regions, refers to a juxtaposition wherein the regions arein a relationship permitting them to function in their intended manner.For example, a control sequence “operably linked” to a coding sequenceis ligated in such a way that expression of the coding sequence isachieved under conditions compatible with the control sequences, such aswhen the appropriate molecules (e.g., inducers and polymerases) arebound to the control or regulatory sequence(s).

The term “polypeptide”, and the terms “protein” and “peptide” which areused interchangeably herein, refers to a polymer of amino acids.Exemplary polypeptides include gene products, naturally-occurringproteins, homologs, orthologs, paralogs, fragments, and otherequivalents, variants and analogs of the foregoing.

The terms “polypeptide fragment” or “fragment”, when used in referenceto a reference polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions mayoccur at the amino-terminus or carboxy-terminus of the referencepolypeptide, or alternatively both. Fragments typically are at least 5,6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20,30, 40 or 50 amino acids long, at least 75 amino acids long, or at least100, 150, 200, 300, 500 or more amino acids long. A fragment can retainone or more of the biological activities of the reference polypeptide.In certain embodiments, a fragment may comprise a domain having thedesired biological activity, and optionally additional amino acids onone or both sides of the domain, which additional amino acids may numberfrom 5, 10, 15, 20, 30, 40, 50, or up to 100 or more residues. Further,fragments can include a sub-fragment of a specific region, whichsub-fragment retains a function of the region from which it is derived.In another embodiment, a fragment may have immunogenic properties.

The term “polypeptide of the invention” refers to a polypeptidecontaining a subject amino acid sequence, or an equivalent or fragmentthereof. Polypeptides of the invention include polypeptides containingall or a portion of a subject amino acid sequence; a subject amino acidsequence with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or moreconservative amino acid substitutions; an amino acid sequence that is atleast 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to asubject amino acid sequence (and every integer between 60 and 100); andfunctional fragments thereof. Polypeptides of the invention also includehomologs, e.g., orthologs and paralogs, of a subject amino acidsequence.

It is also possible to modify the structure of the polypeptides of theinvention for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life, resistance toproteolytic degradation in vivo, etc.). Such modified polypeptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, are considered “functional equivalents” of thepolypeptides described in more detail herein. Such modified polypeptidesmay be produced, for instance, by amino acid substitution, deletion, oraddition, which substitutions may consist in whole or part byconservative amino acid substitutions.

For instance, it is reasonable to expect that an isolated conservativeamino acid substitution, such as replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, will not have a major affect on the biological activity of theresulting molecule. Whether a change in the amino acid sequence of apolypeptide results in a functional homolog may be readily determined byassessing the ability of the variant polypeptide to produce a responsesimilar to that of the wild-type protein. Polypeptides in which morethan one replacement has taken place may readily be tested in the samemanner.

The term “purified” refers to an object species that is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition). A “purified fraction” is acomposition wherein the object species is at least about 50 percent (ona molar basis) of all species present. In making the determination ofthe purity or a species in solution or dispersion, the solvent or matrixin which the species is dissolved or dispersed is usually not includedin such determination; instead, only the species (including the one ofinterest) dissolved or dispersed are taken into account. Generally, apurified composition will have one species that is more than about 80%of all species present in the composition, more than about 85%, 90%,95%, 99% or more of all species present. The object species may bepurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition is essentially a single species. A skilled artisan maypurify a polypeptide of the invention using standard techniques forprotein purification in light of the teachings herein. Purity of apolypeptide may be determined by a number of methods known to those ofskill in the art, including for example, amino-terminal amino acidsequence analysis, gel electrophoresis, mass-spectrometry analysis andthe methods described herein.

The terms “recombinant protein” or “recombinant polypeptide” refer to apolypeptide which is produced by recombinant DNA techniques. An exampleof such techniques includes the case when DNA encoding the expressedprotein is inserted into a suitable expression vector which is in turnused to transform a host cell to produce the protein or polypeptideencoded by the DNA.

The term “regulatory sequence” is a generic term used throughout thespecification to refer to polynucleotide sequences, such as initiationsignals, enhancers, regulators and promoters, that are necessary ordesirable to affect the expression of coding and non-coding sequences towhich they are operably linked. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990), and include, for example, theearly and late promoters of SV40, adenovirus or cytomegalovirusimmediate early promoter, the lac system, the trp system, the TAC or TRCsystem, T7 promoter whose expression is directed by T7 RNA polymerase,the major operator and promoter regions of phage lambda, the controlregions for fd coat protein, the promoter for 3-phosphoglycerate kinaseor other glycolytic enzymes, the promoters of acid phosphatase (e.g.,Pho5), the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. The nature and use of suchcontrol sequences may differ depending upon the host organism. Inprokaryotes, such regulatory sequences generally include promoter,ribosomal binding site, and transcription termination sequences. Theterm “regulatory sequence” is intended to include, at a minimum,components whose presence may influence expression, and may also includeadditional components whose presence is advantageous, for example,leader sequences and fusion partner sequences. In certain embodiments,transcription of a polynucleotide sequence is under the control of apromoter sequence (or other regulatory sequence) which controls theexpression of the polynucleotide in a cell-type in which expression isintended. It will also be understood that the polynucleotide can beunder the control of regulatory sequences which are the same ordifferent from those sequences which control expression of thenaturally-occurring form of the polynucleotide.

The term “sequence homology” refers to the proportion of base matchesbetween two nucleic acid sequences or the proportion of amino acidmatches between two amino acid sequences. When sequence homology isexpressed as a percentage, e.g., 50%, the percentage denotes theproportion of matches over the length of sequence from a desiredsequence that is compared to some other sequence. Gaps (in either of thetwo sequences) are permitted to maximize matching; gap lengths of 15bases or less are usually used, 6 bases or less are used morefrequently, with 2 bases or less used even more frequently. The term“sequence identity” means that sequences are identical (i.e., on anucleotide-by-nucleotide basis for nucleic acids or amino acid-by-aminoacid basis for polypeptides) over a window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the comparison window, determining thenumber of positions at which the identical amino acids or nucleotidesoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window, and multiplying the result by 100 toyield the percentage of sequence identity. Methods to calculate sequenceidentity are known to those of skill in the art and described in furtherdetail below.

The term “soluble” as used herein with reference to a polypeptide of theinvention or other protein, means that upon expression in cell culture,at least some portion of the polypeptide or protein expressed remains inthe cytoplasmic fraction of the cell and does not fractionate with thecellular debris upon lysis and centrifugation of the lysate. Solubilityof a polypeptide may be increased by a variety of art recognizedmethods, including fusion to a heterologous amino acid sequence,deletion of amino acid residues, amino acid substitution (e.g.,enriching the sequence with amino acid residues having hydrophilic sidechains), and chemical modification (e.g., addition of hydrophilicgroups).

The solubility of polypeptides may be measured using a variety of artrecognized techniques, including, dynamic light scattering to determineaggregation state, UV absorption, centrifugation to separate aggregatedfrom non-aggregated material, and SDS gel electrophoresis (e.g., theamount of protein in the soluble fraction is compared to the amount ofprotein in the soluble and insoluble fractions combined). When expressedin a host cell, the polypeptides of the invention may be at least about1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more soluble,e.g., at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or more of the total amount of protein expressed in the cell isfound in the cytoplasmic fraction. In certain embodiments, a one literculture of cells expressing a polypeptide of the invention will produceat least about 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 milligrams ofmore of soluble protein. In an exemplary embodiment, a polypeptide ofthe invention is at least about 10% soluble and will produce at leastabout 1 milligram of protein from a one liter cell culture.

The term “specifically hybridizes” refers to detectable and specificnucleic acid binding. Polynucleotides, oligonucleotides and nucleicacids of the invention selectively hybridize to nucleic acid strandsunder hybridization and wash conditions that minimize appreciableamounts of detectable binding to nonspecific nucleic acids. Stringentconditions may be used to achieve selective hybridization conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence identity between the polynucleotides, oligonucleotides, andnucleic acids of the invention and a nucleic acid sequence of interestwill be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%,or more (and every integer between 30 and 100). In certain instances,hybridization and washing conditions are performed under stringentconditions according to conventional hybridization procedures and asdescribed further herein.

The terms “stringent conditions” or “stringent hybridization conditions”refer to conditions which promote specific hybridization between twocomplementary polynucleotide strands so as to form a duplex. Stringentconditions may be selected to be about 5° C. lower than the thermalmelting point (Tm) for a given polynucleotide duplex at a defined ionicstrength and pH. The length of the complementary polynucleotide strandsand their GC content will determine the Tm of the duplex, and thus thehybridization conditions necessary for obtaining a desired specificityof hybridization. The Tm is the temperature (under defined ionicstrength and pH) at which 50% of a polynucleotide sequence hybridizes toa perfectly matched complementary strand. In certain cases it may bedesirable to increase the stringency of the hybridization conditions tobe about equal to the Tm for a particular duplex.

A variety of techniques for estimating the Tm are available. Typically,G-C base pairs in a duplex are estimated to contribute about 3° C. tothe Tm, while A-T base pairs are estimated to contribute about 2° C., upto a theoretical maximum of about 80-100° C.

However, more sophisticated models of Tm are available in which G-Cstacking interactions, solvent effects, the desired assay temperatureand the like are taken into account. For example, probes can be designedto have a dissociation temperature (Td) of approximately 60° C., usingthe formula: Td=(((3×#GC)+(2×#AT))×37)−562)/#bp)−5; where #GC, #AT, and#bp are the number of guanine-cytosine base pairs, the number ofadenine-thymine base pairs, and the number of total base pairs,respectively, involved in the formation of the duplex.

Hybridization may be carried out in 5×SSC, 4×SSC, 3×SSC, 2×SSC, 1×SSC or0.2×SSC for at least about 1 hour, 2 hours, 5 hours, 12 hours, or 24hours. The temperature of the hybridization may be increased to adjustthe stringency of the reaction, for example, from about 25° C. (roomtemperature), to about 45° C., 50° C., 55° C., 60° C., or 65° C. Thehybridization reaction may also include another agent affecting thestringency, for example, hybridization conducted in the presence of 50%formamide increases the stringency of hybridization at a definedtemperature.

The hybridization reaction may be followed by a single wash step, or twoor more wash steps, which may be at the same or a different salinity andtemperature. For example, the temperature of the wash may be increasedto adjust the stringency from about 25° C. (room temperature), to about45° C., 50° C., 55° C., 60° C., 65° C., or higher. The wash step may beconducted in the presence of a detergent, e.g., 0.1 or 0.2% SDS. Forexample, hybridization may be followed by two wash steps at 65° C. eachfor about 20 minutes in 2×SSC, 0.1% SDS, and optionally two additionalwash steps at 65° C. each for about 20 minutes in 0.2×SSC, 0.1% SDS.

Exemplary stringent hybridization conditions include overnighthybridization at 65° C. in a solution containing 50% formamide,10×Denhardt (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serumalbumin) and 200 μg/ml of denatured carrier DNA, e.g., sheared salmonsperm DNA, followed by two wash steps at 65° C. each for about 20minutes in 2×SSC, 0.1% SDS, and two wash steps at 65° C. each for about20 minutes in 0.2×SSC, 0.1% SDS.

Hybridization may consist of hybridizing two nucleic acids in solution,or a nucleic acid in solution to a nucleic acid attached to a solidsupport, e.g., a filter. When one nucleic acid is on a solid support, aprehybridization step may be conducted prior to hybridization.Prehybridization may be carried out for at least about 1 hour, 3 hoursor 10 hours in the same solution and at the same temperature as thehybridization solution (without the complementary polynucleotidestrand).

Appropriate stringency conditions are known to those skilled in the artor may be determined experimentally by the skilled artisan. See, forexample, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-12.3.6; Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, N.Y.; S. Agrawal (ed.)Methods in Molecular Biology, volume 20; Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization WithNucleic Acid Probes, e.g., part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,N.Y.; and Tibanyenda, N. et al., Eur. J. Biochem. 139:19 (1984) andEbel, S. et al., Biochem. 31:12083 (1992).

The term “vector” refers to a nucleic acid capable of transportinganother nucleic acid to which it has been linked. One type of vectorwhich may be used in accord with the invention is an episome, i.e., anucleic acid capable of extra-chromosomal replication. Other vectorsinclude those capable of autonomous replication and expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer to circular double stranded DNA molecules which,in their vector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

The nucleic acids of the invention may be used as diagnostic reagents todetect the presence or absence of the target DNA or RNA sequences towhich they specifically bind, such as for determining the level ofexpression of a nucleic acid of the invention. In one aspect, thepresent invention contemplates a method for detecting the presence of anucleic acid of the invention or a portion thereof in a sample, themethod of the steps of: (a) providing an oligonucleotide at least eightnucleotides in length, the oligonucleotide being complementary to aportion of a nucleic acid of the invention; (b) contacting theoligonucleotide with a sample containing at least one nucleic acid underconditions that permit hybridization of the oligonucleotide with anucleic acid of the invention or a portion thereof; and (c) detectinghybridization of the oligonucleotide to a nucleic acid in the sample,thereby detecting the presence of a nucleic acid of the invention or aportion thereof in the sample. In another aspect, the present inventioncontemplates a method for detecting the presence of a nucleic acid ofthe invention or a portion thereof in a sample, by (a) providing a pairof single stranded oligonucleotides, each of which is at least eightnucleotides in length, complementary to sequences of a nucleic acid ofthe invention, and wherein the sequences to which the oligonucleotidesare complementary are at least ten nucleotides apart; and (b) contactingthe oligonucleotides with a sample containing at least one nucleic acidunder hybridization conditions; (c) amplifying the nucleotide sequencebetween the two oligonucleotide primers; and (d) detecting the presenceof the amplified sequence, thereby detecting the presence of a nucleicacid of the invention or a portion thereof in the sample.

In another aspect of the invention, the polynucleotide of the inventionis provided in an expression vector containing a nucleotide sequenceencoding a polypeptide of the invention and operably linked to at leastone regulatory sequence. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. The vector's copy number, the ability to control that copynumber and the expression of any other protein encoded by the vector,such as antibiotic markers, should be considered.

An expression vector containing the polynucleotide of the invention canthen be used as a pharmaceutical agent to treat an animal infected withB. hyodysenteriae or as a vaccine (also a pharmaceutical agent) toprevent an animal from being infected with B. hyodysenteriae, or toreduce the symptoms and course of the disease if the animal does becomeinfected. One manner of using an expression vector as a pharmaceuticalagent is to administer a nucleic acid vaccine to the animal at risk ofbeing infected or to the animal after being infected. Nucleic acidvaccine technology is well-described in the art. Some descriptions canbe found in U.S. Pat. No. 6,562,376 (Hooper et al.); U.S. Pat. No.5,589,466 (Felgner, et al.); U.S. Pat. No. 6,673,776 (Felgner, et al.);and U.S. Pat. No. 6,710,035 (Felgner, et al.). Nucleic acid vaccines canbe injected into muscle or intradermally, can be electroporated into theanimal (see WO 01/23537, King et al.; and WO 01/68889, Malone et al.),via lipid compositions (see U.S. Pat. No. 5,703,055, Felgner, et al), orother mechanisms known in the art field.

Expression vectors can also be transfected into bacteria which can beadministered to the target animal to induce an immune response to theprotein encoded by the nucleotides of this invention contained on theexpression vector. The expression vector can contain eukaryoticexpression sequences such that the nucleotides of this invention aretranscribed and translated in the host animal. Alternatively, theexpression vector can be transcribed in the bacteria and then translatedin the host animal. The bacteria used as a carrier of the expressionvector should be attenuated but still invasive. One can use Shigellaspp., Salmonella spp., Escherichia spp., and Aeromonas spp., just toname a few, that have been attenuated but still invasive. Examples ofthese methods can be found in U.S. Pat. No. 5,824,538 (Branstrom et al);U.S. Pat. No. 5,877,159 (Powell, et al.); U.S. Pat. No. 6,150,170(Powell, et al.); U.S. Pat. No. 6,500,419 (Hone, et al.); and U.S. Pat.No. 6,682,729 (Powell, et al.).

Alternatively, the polynucleotides of this invention can be placed incertain viruses which act a vector. Viral vectors can either express theproteins of this invention on the surface of the virus, or carrypolynucleotides of this invention into an animal cell where thepolynucleotide is transcribed and translated into a protein. The animalinfected with the viral vectors can develop an immune response to theproteins encoded by the polynucleotides of this invention. Thereby onecan alleviate or prevent an infection by B. hyodysenteriae in the animalwhich received the viral vectors. Examples of viral vectors can be foundU.S. Pat. No. 5,283,191 (Morgan et al.); U.S. Pat. No. 5,554,525(Sondermeijer et al) and U.S. Pat. No. 5,712,118 (Murphy).

The polynucleotide of the invention may be used to cause expression andover-expression of a polypeptide of the invention in cells propagated inculture, e.g. to produce proteins or polypeptides, including fusionproteins or polypeptides.

This invention pertains to a host cell transfected with a recombinantgene in order to express a polypeptide of the invention. The host cellmay be any prokaryotic or eukaryotic cell. For example, a polypeptide ofthe invention may be expressed in bacterial cells, such as E. coli,insect cells (baculovirus), yeast, plant, or mammalian cells. In thoseinstances when the host cell is human, it may or may not be in a livesubject. Other suitable host cells are known to those skilled in theart. Additionally, the host cell may be supplemented with tRNA moleculesnot typically found in the host so as to optimize expression of thepolypeptide. Alternatively, the nucleotide sequence may be altered tooptimize expression in the host cell, yet the protein produced wouldhave high homology to the originally encoded protein. Other methodssuitable for maximizing expression of the polypeptide will be known tothose in the art.

The present invention further pertains to methods of producing thepolypeptides of the invention. For example, a host cell transfected withan expression vector encoding a polypeptide of the invention may becultured under appropriate conditions to allow expression of thepolypeptide to occur. The polypeptide may be secreted and isolated froma mixture of cells and medium containing the polypeptide. Alternatively,the polypeptide may be retained cytoplasmically and the cells harvested,lysed and the protein isolated.

A cell culture includes host cells, media and other byproducts. Suitablemedia for cell culture are well known in the art. The polypeptide may beisolated from cell culture medium, host cells, or both using techniquesknown in the art for purifying proteins, including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for particular epitopes of a polypeptide of the invention.

Thus, a nucleotide sequence encoding all or a selected portion ofpolypeptide of the invention, may be used to produce a recombinant formof the protein via microbial or eukaryotic cellular processes. Ligatingthe sequence into a polynucleotide construct, such as an expressionvector, and transforming or transfecting into hosts, either eukaryotic(yeast, avian, insect or mammalian) or prokaryotic (bacterial cells),are standard procedures. Similar procedures, or modifications thereof,may be employed to prepare recombinant polypeptides of the invention bymicrobial means or tissue-culture technology.

Suitable vectors for the expression of a polypeptide of the inventioninclude plasmids of the types: pTrcHis-derived plasmids, pET-derivedplasmids, pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derivedplasmids, pBTac-derived plasmids and pUC-derived plasmids for expressionin prokaryotic cells, such as E. coli. The various methods employed inthe preparation of the plasmids and transformation of host organisms arewell known in the art. For other suitable expression systems for bothprokaryotic and eukaryotic cells, as well as general recombinantprocedures, see Molecular Cloning, A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press,1989) Chapters 16 and 17.

Coding sequences for a polypeptide of interest may be incorporated as apart of a fusion gene including a nucleotide sequence encoding adifferent polypeptide. The present invention contemplates an isolatedpolynucleotide containing a nucleic acid of the invention and at leastone heterologous sequence encoding a heterologous peptide linked inframe to the nucleotide sequence of the nucleic acid of the invention soas to encode a fusion protein containing the heterologous polypeptide.The heterologous polypeptide may be fused to (a) the C-terminus of thepolypeptide of the invention, (b) the N-terminus of the polypeptide ofthe invention, or (c) the C-terminus and the N-terminus of thepolypeptide of the invention. In certain instances, the heterologoussequence encodes a polypeptide permitting the detection, isolation,solubilization and/or stabilization of the polypeptide to which it isfused. In still other embodiments, the heterologous sequence encodes apolypeptide such as a poly His tag, myc, HA, GST, protein A, protein G,calmodulin-binding peptide, thioredoxin, maltose-binding protein, polyarginine, poly His-Asp, FLAG, a portion of an immunoglobulin protein,and a transcytosis peptide.

Fusion expression systems can be useful when it is desirable to producean immunogenic fragment of a polypeptide of the invention. For example,the VP6 capsid protein of rotavirus may be used as an immunologiccarrier protein for portions of polypeptide, either in the monomericform or in the form of a viral particle. The nucleic acid sequencescorresponding to the portion of a polypeptide of the invention to whichantibodies are to be raised may be incorporated into a fusion geneconstruct which includes coding sequences for a late vaccinia virusstructural protein to produce a set of recombinant viruses expressingfusion proteins comprising a portion of the protein as part of thevirion. The Hepatitis B surface antigen may also be utilized in thisrole as well. Similarly, chimeric constructs coding for fusion proteinscontaining a portion of a polypeptide of the invention and thepoliovirus capsid protein may be created to enhance immunogenicity (see,for example, EP Publication NO: 0259149; and Evans et al., (1989) Nature339:385; Huang et al., (1988) J. Virol. 62:3855; and Schlienger et al.,(1992) J. Virol. 66:2).

Fusion proteins may facilitate the expression and/or purification ofproteins. For example, a polypeptide of the invention may be generatedas a glutathione-S-transferase (GST) fusion protein. Such GST fusionproteins may be used to simplify purification of a polypeptide of theinvention, such as through the use of glutathione-derivatized matrices(see, for example, Current Protocols in Molecular Biology, eds. Ausubelet al., (N.Y.: John Wiley & Sons, 1991)). In another embodiment, afusion gene coding for a purification leader sequence, such as apoly-(His)/enterokinase cleavage site sequence at the N-terminus of thedesired portion of the recombinant protein, may allow purification ofthe expressed fusion protein by affinity chromatography using a Ni²⁺metal resin. The purification leader sequence may then be subsequentlyremoved by treatment with enterokinase to provide the purified protein(e.g., see Hochuli et al., (1987) J. Chromatography 411: 177; andJanknecht et al., PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene may be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments may be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which maysubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

In other embodiments, the invention provides for nucleic acids of theinvention immobilized onto a solid surface, including, plates,microtiter plates, slides, beads, particles, spheres, films, strands,precipitates, gels, sheets, tubing, containers, capillaries, pads,slices, etc. The nucleic acids of the invention may be immobilized ontoa chip as part of an array. The array may contain one or morepolynucleotides of the invention as described herein. In one embodiment,the chip contains one or more polynucleotides of the invention as partof an array of polynucleotide sequences from the same pathogenic speciesas such polynucleotide(s).

In a preferred form of the invention there is provided isolated B.hyodysenteriae polypeptides as herein described, and also thepolynucleotide sequences encoding these polypeptides. More desirably theB. hyodysenteriae polypeptides are provided in substantially purifiedform.

Preferred polypeptides of the invention will have one or more biologicalproperties (e.g., in vivo, in vitro or immunological properties) of thenative full-length polypeptide. Non-functional polypeptides are alsoincluded within the scope of the invention because they may be useful,for example, as antagonists of the functional polypeptides. Thebiological properties of analogues, fragments, or derivatives relativeto wild type may be determined, for example, by means of biologicalassays.

Polypeptides, including analogues, fragments and derivatives, can beprepared synthetically (e.g., using the well known techniques of solidphase or solution phase peptide synthesis). Preferably, solid phasesynthetic techniques are employed. Alternatively, the polypeptides ofthe invention can be prepared using well known genetic engineeringtechniques, as described infra. In yet another embodiment, thepolypeptides can be purified (e.g., by immunoaffinity purification) froma biological fluid, such as but not limited to plasma, faeces, serum, orurine from animals, including, but not limited to, pig, chicken, goose,duck, turkey, parakeet, human, monkey, dog, cat, horse, hamster, gerbil,rabbit, ferret, horse, cattle, and sheep. An animal can be any mammal orbird.

The B. hyodysenteriae polypeptide analogues include those polypeptideshaving the amino acid sequence, wherein one or more of the amino acidsare substituted with another amino acid which substitutions do notsubstantially alter the biological activity of the molecule.

According to the invention, the polypeptides of the invention producedrecombinantly or by chemical synthesis and fragments or otherderivatives or analogues thereof, including fusion proteins, may be usedas an immunogen to generate antibodies that recognize the polypeptides.

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenic aminoacid sequence contains at least about 5, and preferably at least about10, amino acids. An antigenic portion of a molecule can be the portionthat is immunodominant for antibody or T cell receptor recognition, orit can be a portion used to generate an antibody to the molecule byconjugating the antigenic portion to a carrier molecule forimmunization. A molecule that is antigenic need not be itselfimmunogenic, i.e., capable of eliciting an immune response without acarrier.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567, as well asantigen binding portions of antibodies, including Fab, F(ab′)₂ and F(v)(including single chain antibodies). Accordingly, the phrase “antibodymolecule” in its various grammatical forms as used herein contemplatesboth an intact immunoglobulin molecule and an immunologically activeportion of an immunoglobulin molecule containing the antibody combiningsite. An “antibody combining site” is that structural portion of anantibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically binds an antigen.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction with mercaptoethanol of thedisulfide bonds linking the two heavy chain portions, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response [Hood et al., inImmunology, p. 384, Second Ed., Benjamin/Cummings, Menlo Park, Calif.(1984)]. Often, a primary challenge with an antigen alone, in theabsence of an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminium hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to the polypeptides of the invention. For theproduction of antibody, various host animals can be immunised byinjection with the polypeptide of the invention, including but notlimited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, apolypeptide of the invention can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a polypeptideof the invention, any technique that provides for the production ofantibody molecules by continuous cell lines in culture may be used.These include but are not limited to the hybridoma technique originallydeveloped by Kohler et al., (1975) Nature, 256:495-497, the triomatechnique, the human B-cell hybridoma technique [Kozbor et al., (1983)Immunology Today, 4:72], and the EBV-hybridoma technique to producehuman monoclonal antibodies [Cole et al., (1985) in MonoclonalAntibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc.]. Immortal,antibody-producing cell lines can be created by techniques other thanfusion, such as direct transformation of B lymphocytes with oncogenicDNA, or transfection with Epstein-Barr virus. See, e.g., U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; and 4,493,890.

In an additional embodiment of the invention, monoclonal antibodies canbe produced in germ-free animals utilising recent technology. Accordingto the invention, chicken or swine antibodies may be used and can beobtained by using chicken or swine hybridomas or by transforming B cellswith EBV virus in vitro. In fact, according to the invention, techniquesdeveloped for the production of “chimeric antibodies” [Morrison et al.,(1984) J. Bacteriol., 159-870; Neuberger et al., (1984) Nature,312:604-608; Takeda et al., (1985) Nature, 314:452-454] by splicing thegenes from a mouse antibody molecule specific for a polypeptide of theinvention together with genes from an antibody molecule of appropriatebiological activity can be used; such antibodies are within the scope ofthis invention. Such chimeric antibodies are preferred for use intherapy of intestinal diseases or disorders (described infra), since theantibodies are much less likely than xenogenic antibodies to induce animmune response, in particular an allergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce single chain antibodies specific for an polypeptide of theinvention. An additional embodiment of the invention utilises thetechniques described for the construction of Fab expression libraries[Huse et al., (1989) Science, 246:1275-1281] to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor a polypeptide of the invention.

Antibody fragments, which contain the idiotype of the antibody molecule,can be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA, “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),Western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays,immunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labelled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention. For example, to select antibodies thatrecognise a specific epitope of a polypeptide of the invention, one mayassay generated hybridomas for a product that binds to a fragment of apolypeptide of the invention containing such epitope.

The invention also covers diagnostic and prognostic methods to detectthe presence of B. hyodysenteriae using a polypeptide of the inventionand/or antibodies which bind to the polypeptide of the invention andkits useful for diagnosis and prognosis of B. hyodysenteriae infections.

Diagnostic and prognostic methods will generally be conducted using abiological sample obtained from an animal, such as chicken or swine. A“sample” refers to an animal's tissue or fluid suspected of containing aBrachyspira species, such as B. hyodysenteriae, or its polynucleotidesor its polypeptides. Examples of such tissue or fluids include, but notlimited to, plasma, serum, fecal material, urine, lung, heart, skeletalmuscle, stomach, intestines, and in vitro cell culture constituents.

The invention provides methods for detecting the presence of apolypeptide of the invention in a sample, with the following steps: (a)contacting a sample suspected of containing a polypeptide of theinvention with an antibody (preferably bound to a solid support) thatspecifically binds to the polypeptide of the invention under conditionswhich allow for the formation of reaction complexes comprising theantibody and the polypeptide of the invention; and (b) detecting theformation of reaction complexes comprising the antibody and polypeptideof the invention in the sample, wherein detection of the formation ofreaction complexes indicates the presence of the polypeptide of theinvention in the sample.

Preferably, the antibody used in this method is derived from anaffinity-purified polyclonal antibody, and more preferably a monoclonalantibody. In addition, it is preferable for the antibody molecules usedherein be in the form of Fab, Fab′, F(ab′)₂ or F(v) portions or wholeantibody molecules.

Particularly preferred methods for detecting B. hyodysenteriae based onthe above method include enzyme linked immunosorbent assays,radioimmunoassays, immunoradiometric assays and immunoenzymatic assays,including sandwich assays using monoclonal and/or polyclonal antibodies.

Three such procedures that are especially useful utilise eitherpolypeptide of the invention (or a fragment thereof) labelled with adetectable label, antibody Ab₁ labelled with a detectable label, orantibody Ab₂ labelled with a detectable label. The procedures may besummarized by the following equations wherein the asterisk indicatesthat the particle is labelled and “AA” stands for the polypeptide of theinvention:AA*+Ab ₁ =AA*Ab ₁  (A.)AA+Ab* ₁ =AAAb ₁*  (B.)AA+Ab ₁ +Ab ₂ *=Ab ₁ AAAb ₂*  (C.)

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilised within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure B isrepresentative of well-known competitive assay techniques. Procedure C,the “sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and4,016,043. Still other procedures are known, such as the “doubleantibody” or “DASP” procedure, and can be used.

In each instance, the polypeptide of the invention form complexes withone or more antibody(ies) or binding partners and one member of thecomplex is labelled with a detectable label. The fact that a complex hasformed and, if desired, the amount thereof, can be determined by knownmethods applicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This reaction is because Ab₁, raised in onemammalian species, has been used in another species as an antigen toraise the antibody, Ab₂. For example, Ab₂ may be raised in goats usingrabbit antibodies as antigens. Ab₂ therefore would be anti-rabbitantibody raised in goats. For purposes of this description and claims,Ab₁ will be referred to as a primary antibody, and Ab₂ will be referredto as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals that fluoresce when exposed to ultravioletlight, and others. Examples of fluorescent materials capable of beingutilised as labels include fluorescein, rhodamine and auramine. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate. Examples ofpreferred isotope include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co,⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. The radioactive label can be detectedby any of the currently available counting procedures. While manyenzymes can be used, examples of preferred enzymes are peroxidase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plus peroxidase and alkaline phosphatase. Enzyme are conjugatedto the selected particle by reaction with bridging molecules such ascarbodiimides, diisocyanates, glutaraldehyde and the like. Enzyme labelscan be detected by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques. U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 arereferred to by way of example for their disclosure of alternatelabelling material and methods.

The invention also provides a method of detecting antibodies to apolypeptide of the invention in biological samples, using the followingsteps: (a) providing a polypeptide of the invention or a fragmentthereof; (b) incubating a biological sample with said polypeptide of theinvention under conditions which allow for the formation of anantibody-antigen complex; and (c) determining whether anantibody-antigen complex with the polypeptide of the invention isformed.

In another embodiment of the invention there are provided in vitromethods for evaluating the level of antibodies to a polypeptide of theinvention in a biological sample using the following steps: (a)detecting the formation of reaction complexes in a biological sampleaccording to the method noted above; and (b) evaluating the amount ofreaction complexes formed, which amount of reaction complexescorresponds to the level of polypeptide of the invention in thebiological sample.

Further there are provided in vitro methods for monitoring therapeutictreatment of a disease associated with B. hyodysenteriae in an animalhost by evaluating, as describe above, the levels of antibodies to apolypeptide of the invention in a series of biological samples obtainedat different time points from an animal host undergoing such therapeutictreatment.

The present invention further provides methods for detecting thepresence or absence of B. hyodysenteriae in a biological sample by: (a)bringing the biological sample into contact with a polynucleotide probeor primer of polynucleotide of the invention under suitable hybridizingconditions; and (b) detecting any duplex formed between the probe orprimer and nucleic acid in the sample.

According to one embodiment of the invention, detection of B.hyodysenteriae may be accomplished by directly amplifying polynucleotidesequences from biological sample, using known techniques and thendetecting the presence of polynucleotide of the invention sequences.

In one form of the invention, the target nucleic acid sequence isamplified by PCR and then detected using any of the specific methodsmentioned above. Other useful diagnostic techniques for detecting thepresence of polynucleotide sequences include, but are not limited to: 1)allele-specific PCR; 2) single stranded conformation analysis; 3)denaturing gradient gel electrophoresis; 4) RNase protection assays; 5)the use of proteins which recognize nucleotide mismatches, such as theE. coli mutS protein; 6) allele-specific oligonucleotides; and 7)fluorescent in situ hybridisation.

In addition to the above methods polynucleotide sequences may bedetected using conventional probe technology. When probes are used todetect the presence of the desired polynucleotide sequences, thebiological sample to be analysed, such as blood or serum, may betreated, if desired, to extract the nucleic acids. The samplepolynucleotide sequences may be prepared in various ways to facilitatedetection of the target sequence; e.g. denaturation, restrictiondigestion, electrophoresis or dot blotting. The targeted region of thesample polynucleotide sequence usually must be at least partiallysingle-stranded to form hybrids with the targeting sequence of theprobe. If the sequence is naturally single-stranded, denaturation willnot be required. However, if the sequence is double-stranded, thesequence will probably need to be denatured. Denaturation can be carriedout by various techniques known in the art.

Sample polynucleotide sequences and probes are incubated underconditions that promote stable hybrid formation of the target sequencein the probe with the putative desired polynucleotide sequence in thesample. Preferably, high stringency conditions are used in order toprevent false positives.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labelled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand that islabelled, either directly or indirectly. Suitable labels and methods forlabelling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labelledmoiety.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention may employ a cocktail ofnucleic acid probes capable of detecting the desired polynucleotidesequences of this invention. Thus, in one example to detect the presenceof polynucleotide sequences of this invention in a cell sample, morethan one probe complementary to a polynucleotide sequences is employedand in particular the number of different probes is alternatively 2, 3,or 5 different nucleic acid probe sequences.

The polynucleotide sequences described herein (preferably in the form ofprobes) may also be immobilised to a solid phase support for thedetection of Brachyspira species, including but not limited to B.hyodysenteriae, B. intermedia, B. alvinipulli, B. aalborgi, B. innocens,B. murdochii, and B. pilosicoli. Alternatively the polynucleotidesequences described herein will form part of a library of DNA moleculesthat may be used to detect simultaneously a number of different genesfrom Brachyspira species, such as B. hyodysenteriae. In a furtheralternate form of the invention polynucleotide sequences describedherein together with other polynucleotide sequences (such as from otherbacteria or viruses) may be immobilised on a solid support in such amanner permitting identification of the presence of a Brachyspiraspecies, such as B. hyodysenteriae and/or any of the otherpolynucleotide sequences bound onto the solid support.

Techniques for producing immobilised libraries of DNA molecules havebeen described in the art. Generally, most prior art methods describethe synthesis of single-stranded nucleic acid molecule libraries, usingfor example masking techniques to build up various permutations ofsequences at the various discrete positions on the solid substrate. U.S.Pat. No. 5,837,832 describes an improved method for producing DNA arraysimmobilised to silicon substrates based on very large scale integrationtechnology. In particular, U.S. Pat. No. 5,837,832 describes a strategycalled “tiling” to synthesize specific sets of probes at spatiallydefined locations on a substrate that may be used to produced theimmobilised DNA libraries of the present invention. U.S. Pat. No.5,837,832 also provides references for earlier techniques that may alsobe used. Thus polynucleotide sequence probes may be synthesised in situon the surface of the substrate.

Alternatively, single-stranded molecules may be synthesised off thesolid substrate and each pre-formed sequence applied to a discreteposition on the solid substrate. For example, polynucleotide sequencesmay be printed directly onto the substrate using robotic devicesequipped with either pins or pizo electric devices.

The library sequences are typically immobilised onto or in discreteregions of a solid substrate. The substrate may be porous to allowimmobilisation within the substrate or substantially non-porous, inwhich case the library sequences are typically immobilised on thesurface of the substrate. The solid substrate may be made of anymaterial to which polypeptides can bind, either directly or indirectly.Examples of suitable solid substrates include flat glass, siliconwafers, mica, ceramics and organic polymers such as plastics, includingpolystyrene and polymethacrylate. It may also be possible to usesemi-permeable membranes such as nitrocellulose or nylon membranes,which are widely available. The semi-permeable membranes may be mountedon a more robust solid surface such as glass. The surfaces mayoptionally be coated with a layer of metal, such as gold, platinum orother transition metal.

Preferably, the solid substrate is generally a material having a rigidor semi-rigid surface. In preferred embodiments, at least one surface ofthe substrate will be substantially flat, although in some embodimentsit may be desirable to physically separate synthesis regions fordifferent polymers with, for example, raised regions or etched trenches.It is also preferred that the solid substrate is suitable for the highdensity application of DNA sequences in discrete areas of typically from50 to 100 μm, giving a density of 10000 to 40000 dots/cm⁻².

The solid substrate is conveniently divided up into sections. This maybe achieved by techniques such as photoetching, or by the application ofhydrophobic inks, for example Teflon®-based inks (Cel-line®, USA).

Discrete positions, in which each different member of the library islocated may have any convenient shape, e.g., circular, rectangular,elliptical, wedge-shaped, etc.

Attachment of the polynucleotide sequences to the substrate may be bycovalent or non-covalent means. The polynucleotide sequences may beattached to the substrate via a layer of molecules to which the librarysequences bind. For example, the polynucleotide sequences may belabelled with biotin and the substrate coated with avidin and/orstreptavidin. A convenient feature of using biotinylated polynucleotidesequences is that the efficiency of coupling to the solid substrate canbe determined easily. Since the polynucleotide sequences may bind onlypoorly to some solid substrates, it is often necessary to provide achemical interface between the solid substrate (such as in the case ofglass) and the nucleic acid sequences. Examples of suitable chemicalinterfaces include hexaethylene glycol. Another example is the use ofpolylysine coated glass, the polylysine then being chemically modifiedusing standard procedures to introduce an affinity ligand. Other methodsfor attaching molecules to the surfaces of solid substrate by the use ofcoupling agents are known in the art, see for example WO98/49557.

Binding of complementary polynucleotide sequences to the immobilisednucleic acid library may be determined by a variety of means such aschanges in the optical characteristics of the bound polynucleotidesequence (i.e. by the use of ethidium bromide) or by the use of labellednucleic acids, such as polypeptides labelled with fluorophores. Otherdetection techniques that do not require the use of labels includeoptical techniques such as optoacoustics, reflectometry, ellipsometryand surface plasmon resonance (see WO97/49989).

Thus, the present invention provides a solid substrate havingimmobilized thereon at least one polynucleotide of the presentinvention, preferably two or more different polynucleotide sequences ofthe present invention.

The present invention also can be used as a prophylactic or therapeutic,which may be utilised for the purpose of stimulating humoral and cellmediated responses in animals, such as chickens and swine, therebyproviding protection against colonisation with Brachyspira species,including but not limited to B. hyodysenteriae, B. suanatina, B.intermedia, B. alvinipulli, B. aalborgi, B. innocens, B. murdochii, andB. pilosicoli. Natural infection with a Brachyspira species, such as B.hyodysenteriae induces circulating antibody titres against the proteinsdescribed herein. Therefore, the amino acid sequences described hereinor parts thereof, have the potential to form the basis of a systemicallyor orally administered prophylactic or therapeutic to provide protectionagainst intestinal spirochaetosis.

Accordingly, in one embodiment the present invention provides the aminoacid sequences described herein or fragments thereof or antibodies thatbind the amino acid sequences or the polynucleotide sequences describedherein in a therapeutically effective amount admixed with apharmaceutically acceptable carrier, diluent, or excipient.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to reduce by at least about 15%, preferably by atleast 50%, more preferably by at least 90%, and most preferably prevent,a clinically significant deficit in the activity, function and responseof the animal host. Alternatively, a therapeutically effective amount issufficient to cause an improvement in a clinically significant conditionin the animal host.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similarly untoward reaction, such as gastricupset and the like, when administered to an animal. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which thecompound is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water or saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in Martin, Remington's Pharmaceutical Sciences, 18th Ed.,Mack Publishing Co., Easton, Pa., (1990).

In a more specific form of the invention there are providedpharmaceutical compositions comprising therapeutically effective amountsof the amino acid sequences described herein or an analogue, fragment orderivative product thereof or antibodies thereto together withpharmaceutically acceptable diluents, preservatives, solubilizes,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength and additives such as detergents and solubilizingagents (e.g., Tween® 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol). The materialmay be incorporated into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycoli.c acid, etc. or into liposomes,Hylauronic acid may also be used. Such compositions may influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of the present proteins and derivatives. See, e.g., Martin,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 that are herein incorporated byreference. The compositions may be prepared in liquid form, or may be indried powder, such as lyophilised form.

Alternatively, the polynucleotides of the invention can be optimized forexpression in plants (e.g., corn). The plant may be transformed withplasmids containing the optimized polynucleotides. Then the plant isgrown, and the proteins of the invention are expressed in the plant, orthe plant-optimized version is expressed. The plant is later harvested,and the section of the plant containing the proteins of the invention isprocessed into feed for the animal. This animal feed will impartimmunity against B. hyodysenteriae when eaten by the animal. Examples ofprior art detailing these methods can be found in U.S. Pat. No.5,914,123 (Arntzen, et al.); U.S. Pat. No. 6,034,298 (Lam, et al.); andU.S. Pat. No. 6,136,320 (Arntzen, et al.).

It will be appreciated that pharmaceutical compositions providedaccordingly to the invention may be administered by any means known inthe art. Preferably, the pharmaceutical compositions for administrationare administered by injection, orally, or by the pulmonary, or nasalroute. The amino acid sequences described herein or antibodies derivedtherefrom are more preferably delivered by intravenous, intraarterial,intraperitoneal, intramuscular, or subcutaneous routes ofadministration. Alternatively, the amino acid sequence described hereinor antibodies derived therefrom, properly formulated, can beadministered by nasal or oral administration.

Also encompassed by the present invention is the use of polynucleotidesequences of the invention, as well as antisense and ribozymepolynucleotide sequences hybridisable to a polynucleotide sequenceencoding an amino acid sequence according to the invention, formanufacture of a medicament for modulation of a disease associated B.hyodysenteriae.

Polynucleotide sequences encoding antisense constructs or ribozymes foruse in therapeutic methods are desirably administered directly as anaked nucleic acid construct. Uptake of naked nucleic acid constructs bybacterial cells is enhanced by several known transfection techniques,for example those including the use of transfection agents. Example ofthese agents include cationic agents (for example calcium phosphate andDEAE-dextran) and lipofectants. Typically, nucleic acid constructs aremixed with the transfection agent to produce a composition.

Alternatively the antisense construct or ribozymes may be combined witha pharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline. Thecomposition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular, oral or transdermaladministration. The routes of administration described are intended onlyas a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and any dosage for anyparticular animal and condition.

The invention also includes kits for screening animals suspected ofbeing infected with a Brachyspira species, such as B. hyodysenteriae orto confirm that an animal is infected with a Brachyspira species, suchas B. hyodysenteriae. In a further embodiment of this invention, kitssuitable for use by a specialist may be prepared to determine thepresence or absence of Brachyspira species, including but not limited toB. hyodysenteriae, B. suanatina, B. intermedia, B. alvinipulli, B.aalborgi, B. innocens, B. murdochii, and B. pilosicoli in suspectedinfected animals or to quantitatively measure a Brachyspira species,including but not limited to B. hyodysenteriae, B. suanatina, B.intermedia, B. alvinipulli, B. aalborgi and B. pilosicoli infection. Inaccordance with the testing techniques discussed above, such kits cancontain at least a labelled version of one of the amino acid sequencesdescribed herein or its binding partner, for instance an antibodyspecific thereto, and directions depending upon the method selected,e.g., “competitive,” “sandwich,” “DASP” and the like. Alternatively,such kits can contain at least a polynucleotide sequence complementaryto a portion of one of the polynucleotide sequences described hereintogether with instructions for its use. The kits may also containperipheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit for the demonstration of the presence of aBrachyspira species, including but not limited to B. hyodysenteriae, B.suanatina, B. intermedia, B. alvinipulli, B. aalborgi, B. innocens, B.murdochii, and B. pilosicoli, may contain the following:

(a) a predetermined amount of at least one labelled immunochemicallyreactive component obtained by the direct or indirect attachment of oneof the amino acid sequences described herein or a specific bindingpartner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may contain:

(a) a known amount of one of the amino acid sequences described hereinas described above (or a binding partner) generally bound to a solidphase to form an immunosorbent, or in the alternative, bound to asuitable tag, or there are a plural of such end products, etc;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may contain:

(a) a labelled component which has been obtained by coupling one of theamino acid sequences described herein to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

-   -   (i) a ligand capable of binding with the labelled component (a);    -   (ii) a ligand capable of binding with a binding partner of the        labelled component (a);    -   (iii) a ligand capable of binding with at least one of the        component(s) to be determined; or    -   (iv) a ligand capable of binding with at least one of the        binding partners of at least one of the component(s) to be        determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between one of the amino acid sequences described herein and aspecific binding partner thereto.

Preparation of Genomic Library

A genomic library is prepared using an Australian porcine field isolateof B. hyodysenteriae (strain WA1), This strain has beenwell-characterised and shown to he virulent following experimentalchallenge of pigs. The cetyltrimetyhylammonium bromide (CTAB) method isused to prepare high quality chromosomal DNA suitable for preparation ofgenomic DNA libraries. B. hyodysenteriae is grown in 100 ml anaerobictrypticase soy broth culture to a cell density of 10⁹ cells/ml. Thecells are harvested at 4,000×g for 10 minutes, and the cell pelletresuspended in 9.5 ml TE buffer. SDS is added to a final concentrationof 0.5% (w/v), and the cells lysed at 37° C. for 1 hour with 100 μg ofProteinase K. NaCl is added to a final concentration of 0.7 M and 1.5 mlCTAB/NaCl solution (10% w/v CTAB, 0.7 M NaCl) is added before incubatingthe solution at 65° C. for 20 minutes. The lysate is extracted with anequal volume of chloroform/isoamyl alcohol, and the tube is centrifugedat 6,000×g for 10 minutes to separate the phases. The aqueous phase istransferred to a fresh tube and 0.6 volumes of isopropanol are added toprecipitate the high molecular weight DNA. The precipitated DNA iscollected using a hooked glass rod and transferred to a tube containing1 ml of 70% (v/v) ethanol. The tube is centrifuged at 10,000×g and thepelleted DNA redissolved in 4 ml TE buffer overnight. A cesium chloridegradient containing 1.05 g/ml CsC1 and 0.5 mg/ml ethidium bromide isprepared using the redissolved DNA solution. The gradient is transferredto 4 ml scalable centrifuge tubes and centrifuged at 70,000×g overnightat 15° C. The separated DNA is visualized under an ultraviolet light,and the high molecular weight DNA is withdrawn from the gradient using a15-g needle. The ethidium bromide is removed from the DNA by sequentialextraction with CsCl-saturated isopropanol. The purified chromosomal DNAis dialysed against 2 liters TE buffer and precipitated withisopropanol. The resuspended genomic DNA is sheared using a GeneMachinesHydroshear™ (Genomic Solutions, Ann Arbor, Mich.), and the sheared DNAis filled-in using Klenow DNA polymerase to generate blunt-end.fragments. One hundred ng of the blunt-end DNA fragments is ligated with25 ng of pSMART® VC vector (Lucigen, Meddleton, Wis.) using CloneSmart®DNA ligase, The ligated DNA is then eleetroporated into E colielectrocompetent cells. A small insert (2-3 kb) library and mediuminsert (3-40 kb) library is constructed into the low copy version of thepSMART® VC vector.

Genomic Sequencing

After the genomic library is obtained, individual clones of E. coilcontaining the pSMART® VC vector are picked. The plasmid DNA is purifiedand sequenced. The purified plasmids are subjected to automated directsequencing of the pSMART® VC vector using the forward and reverseprimers specific for the pSMART® VC vector. Each sequencing reaction isperformed in a 10 μl volume consisting of 200 ng of plasmid DNA, 2 pmolof primer, and 4 μl of the ABI PRISM™ BigDye® Terminator CycleSequencing Ready Reaction Mix (PE Applied Biosystems, Foster City,Calif.). Cycling conditions involve a 2 minute denaturing step at 96°C., followed by 25 cycles of denaturation at 96° C. for 10 seconds, anda combined primer annealing and extension step at 60° C. for 4 minutes.Residual dye terminators are removed from the sequencing products byprecipitation with 95% (v/v) ethanol containing 85 mM sodium acetate (pH5.2), 3 mM EDTA (pH 8), and vacuum dried. The plasmids are sequenced induplicate using each primer. Sequencing products are analysed using anABI 373A DNA Sequencer (PE Applied Biosystems).

Annotation

Partial genome sequences for B. hyodysenteriae are assembled andannotated using a range of public domain bioinformatics tools to analyseand re-analyse the sequences as part of a quality assurance procedure ondata analysis. Open reading frames (ORFs) are predicted using a varietyof programs including GeneMark™, GLIMMER, ORPHEUS, SELFID and GetORF,Putative ORFS are examined for homology (DNA and protein) with existinginternational databases using searches including BLAST and FASTA. Allthe predicted ORFs are analysed to determine their cellular localisationusing programs including PSI-BLAST, FASTA, MOTIFS, FINDPATTERNS, PHD,SIGNALP and PSORT. Databases including Interpro, Prosite, ProDom, Pfamand Blocks are used to predict surface associated proteins such astransmembrane domains, leader peptides, homologies to known surfaceproteins, lipoprotein signature, outer membrane anchoring motifs andhost cell binding domains. Phylogenetic and other molecular evolutionanalysis is conducted with the identified genes and with other speciesto assist in the assignment of function. The in silico analysis of bothpartially sequenced genomes produces a comprehensive list of all thepredicted ORFS present in the sequence data available. Each ORF isinterrogated for descriptive information such as predicted molecularweight, isoelectric point, hydrophobicity, and subcellular localisationto enable correlation with the in vitro properties of the native geneproduct. Predicted genes which encode proteins similar to surfacelocalized components and/or virulence factors in other pathogenicbacteria are selected as potential vaccine targets.

Bioinformatics Results

The shotgun sequencing of the B. hyodysenteriae genome results in 94.6%(3028.6 kb out of a predicted 3200 kb) of the genome being sequenced.The B. hyodysenteriae sequence is comprised of 294 contigs with anaverage contig size of 10.3 kb. For B. hyodysenteriae, 2,593open-reading frames (ORFs) are predicted from the 294 contigs.Comparison of the predicted ORFs with genes present in the nucleic acidand protein databases indicate that approximately 70% of the ORFs havehomology with genes contained in the public databases. The remaining 30%of the predicted ORFs have no known identity.

Vaccine Candidates

To help reduce the number of ORFs that would be tested as a vaccinecandidate, ORF's showing reasonable homology (E-value less than e⁻¹⁵)with outer surface proteins, secreted proteins, and possible virulencefactors present in public databases are selected as potential vaccinecandidates. Of the 2,593 ORFs obtained in the genomic shotgunsequencing, many passed this test but the results of thirty-three genesare presented here. Table 1 shows thirty-three genes selected aspotential vaccine targets and their similarity with other known aminoacid sequences obtained from SWISS-PROT database. It is noted that thepercent identity of amino acids does not raise above 58% while thepercent similarity or homology of amino acids remains less than 71%,thus indicating that these ORFs are unique.

TABLE 1 Identity of Protein Identity Similarity Accession Gene WithHighest Homology (amino acids) (amino acids) Number NAV-H54 Variablesurface protein (VspD) of 106/223  136/223 O68157 Brachyspirahyodysenteriae (47%) (60%) NAV-H55 Flagellar protein B of 58/213 108/213Q72SJ3 Leptospira interrogans (27%) (50%) NAV-H56 Myosin-like majorantigen 288/1509  609/1509 P21249 (19%) (40%) NAV-H57 Lytic mureintransglycosylate (possibly 97/318 158/318 Q6F7W9 outer membrane-bound)(25%) (41%) NAV-H58 Outer membrane protein of 57/175  90/175 P96127Treponema pallidum (32%) (51%) NAV-H59 Myosin-like major antigen204/1012  432/1012 P21249 (20%) (42%) NAV-H60 Outer membrane protein andrelated 46/139  68/139 COG2885 peptidoglycan-associated lipoprotein(33%) (48%) NAV-H61 Putative lipoprotein of 336/805  483/805 Q73NT0Treponema denticola (41%) (60%) NAV-H62 NlpA lipoprotein of 92/269151/269 Q303L3 Streptococcus suis (34%) (56%) NAV-H63 NlpA lipoproteinof 106/312  167/312 Q303L3 Streptococcus suis (33%) (53%) NAV-H64 NlpAlipoprotein of 112/337  198/337 Q303L3 Streptococcus suis (33%) (58%)NAV-H65 Putative secreted protein of 50/127  68/127 Q849M9 Streptomycesviolaceoruber (39%) (53%) NAV-H66 Toxin (YoeB) of Escherichia coli42/85  60/85 P69349 (58%) (76%) NAV-H67 Outer membrane protein (TolC)80/350 171/350 Q2Z054 (20%) (39%) NAV-H68 Probable hemolysin-relatedprotein of 121/415  204/415 Q3ZYX1 Dehalococcoides sp. (29%) (49%)NAV-H69 Outer surface protein of 193/357  256/357 Q4MWS0 Bacillus cereus(54%) (71%) NAV-H70 Membrane associated lipoprotein of 146/417  203/417Q2SRL9 Vibrio vulnificus (35%) (48%) NAV-H71 Surface layer protein of72/201 112/201 Q8TJE3 Methanosarcina barkeri (36%) (49%) NAV-H72 Lyticmurein transglycosylate (possibly 51/141  6/141 P44049 outermembrane-bound) (36%) (53%) NAV-H73 Toxin (YoeB) of Escherichia coli42/84  62/84 P69349 (50%) (73%) NAV-H74 Outer membrane protein/ 210/833 339/833 COG4775 protective antigen (25%) (40%) NAV-H22 variable surfaceprotein (VspH) of 29% 43% AAK14803.1 Brachyspira hyodysenteriae(133/454)  (199/454) NAV-H23 membrane associated lipoprotein of 43% 60%AAF27178.1 Mycoplasma mycoides (114/263)  (159/263) NAV-H24 Outermembrane lipoprotein of 32% 53% ZP00300921.1 Geobacter metallireducens(46/142)  (76/142) NAV-H30 surface antigen (BspA) of 38% 55% AAC82625.1Bacteroides forsythus (83/216) (120/216) NAV-H32 hemolytic protein(HlpA) of Nostoc sp. 35% 56% NP488469.1 (49/137)  (77/137) NAV-H33hemolytic protein of 54% 70% AAC05836.1 Prevotella intermedia (64/117) (83/117) NAV-H37 virulence-mediating protein (VirC) of 36% 63%NP800579.1 Vibrio parahaemolyticus (58/159) (101/159) NAV-H40 lyticmurein transglycosylase (contains 26% 41% ZP00146104.1 LysM/invasindomains) (120/449)  (185/449) NAV-H41 surface antigen BspA of 41% 57%AAC82625.1 Bacteroides forsythus (84/201) (115/201) NAV-H43 Hemolysinsand related proteins of 35% 56% ZP00162711.2 Anabaena variabilis(150/425)  (242/425) NAV-H44 outer membrane porin of 20% 41% YP001419.1Leptospira interrogans (79/393) (163/393) NAV-H45 virulence factor(MviN) protein of 32% 49% NP952225.1 Geobacter sulfurreducens (153/469) (231/469)

The DNA and amino acid sequences of NAV-H54 are found in SEQ ID NOs: 1and 2, respectively. The DNA and amino acid sequences of NAV-H55 arefound in SEQ ID NOs: 3 and 4, respectively. The DNA and amino acidsequences of NAV-H56 are found in SEQ ID NOs: 5 and 6, respectively. TheDNA and amino acid sequences of NAV-H57 are found in SEQ ID NOs: 7 and8, respectively. The DNA and amino acid sequences of NAV-H58 are foundin SEQ ID NOs: 9 and 10, respectively. The DNA and amino acid sequencesof NAV-H59 are found in SEQ ID NOs: 11 and 12, respectively. The DNA andamino acid sequences of NAV-H60 are found in SEQ ID NOs: 13 and 14,respectively. The DNA and amino acid sequences of NAV-H61 are found inSEQ ID NOs: 15 and 16, respectively. The DNA and amino acid sequences ofNAV-H62 are found in SEQ ID NOs: 17 and 18, respectively. The DNA andamino acid sequences of NAV-H63 are found in SEQ ID NOs: 19 and 20,respectively. The DNA and amino acid sequences of NAV-H64 are found inSEQ ID NOs: 21 and 22, respectively. The DNA and amino acid sequences ofNAV-H65 are found in SEQ ID NOs: 23 and 24, respectively. The DNA andamino acid sequences of NAV-H66 are found in SEQ ID NOs: 25 and 26,respectively. The DNA and amino acid sequences of NAV-H67 are found inSEQ ID NOs: 27 and 28, respectively. The DNA and amino acid sequences ofNAV-H68 are found in SEQ ID NOs: 29 and 30, respectively. The DNA andamino acid sequences of NAV-H69 are found in SEQ ID NOs: 31 and 32,respectively. The DNA and amino acid sequences of NAV-H70 are found inSEQ ID NOs: 33 and 34, respectively. The DNA and amino acid sequences ofNAV-H71 are found in SEQ ID NOs: 35 and 36, respectively. The DNA andamino acid sequences of NAV-H72 are found in SEQ ID NOs: 37 and 38,respectively. The DNA and amino acid sequences of NAV-H73 are found inSEQ ID NOs: 39 and 40, respectively. The DNA and amino acid sequences ofNAV-H74 are found in SEQ ID NOs: 41 and 42, respectively.

The DNA and amino acid sequences of NAV-H22 are found in SEQ ID NOs: 43and 44, respectively. The DNA and amino acid sequences of NAV-H23 arefound in SEQ ID NOs: 45 and 46, respectively. The DNA and amino acidsequences of NAV-H24 are found in SEQ ID NOs: 47 and 48, respectively.The DNA and amino acid sequences of NAV-H30 are found in SEQ ID NOs: 49and 50, respectively. The DNA and amino acid sequences of NAV-H32 arefound in SEQ ID NOs: 51 and 52, respectively. The DNA and amino acidsequences of NAV-H33 are found in SEQ ID NOs: 53 and 54, respectively.The DNA and amino acid sequences of NAV-H37 are found in SEQ ID NOs: 55and 56, respectively. The DNA and amino acid sequences of NAV-H40 arefound in SEQ ID NOs: 57 and 58, respectively. The DNA and amino acidsequences of NAV-H41 are found in SEQ ID NOs: 59 and 60, respectively.The DNA and amino acid sequences of NAV-H43 are found in SEQ ID NOs: 61and 62, respectively. The DNA and amino acid sequences of NAV-H44 arefound in SEQ ID NOs: 63 and 64, respectively. The DNA and amino acidsequences of NAV-H45 are found in SEQ ID NOs: 65 and 66, respectively.

To further reduce the number of ORFs that would be tested as a vaccinecandidate, gene products predicted by the in silico analysis to belocalised in the cytoplasm or inner membrane of the spirochaete areabandoned. As a result, twenty one of the thirty three genes presentedin Table 1 are further analysed. These include NAV-H58, NAV-H60,NAV-H62, NAV-H64, NAV-H66, NAV-H67, NAV-H69, NAV-H71, NAV-H73, NAV-H22,NAV-H23, NAV-H24, NAV-H30, NAV-H32, NAV-H33, NAV-H37, NAV-H40, NAV-H41,NAV-H43, NAV-H44, and NAV-H45.

Analysis of Gene Distribution Using Polymerase Chain Reaction (PCR)

One or two primer pairs which anneal to different regions of the targetgene encoding region are designed and optimised for PCR detection.Individual primers are designed using Oligo Explorer 1.2 and primer setswith calculated melting temperatures of approximately 55-60° C. areselected. These primers sets are also selected to generate PCR productsgreater than 200 bp. A medium-stringency primer annealing temperature of50° C. is selected for the distribution analysis PCR. Themedium-stringency conditions would allow potential minor mismatchedsequences (because of strain differences) occurring at the primerbinding sites to not affect primer binding. Distribution analysis of thetwenty one B. hyodysenteriae target genes are performed on 23 strains ofB. hyodysenteriae, including two strains which have been shown to beavirulent. PCR analysis is performed in a 25 μl total volume using TaqDNA polymerase (Biotech International, Thurmont, Md.). The amplificationmixture consists of 1×PCR buffer (containing 1.5 mM of MgCl₂), 1 U ofTaq DNA polymerase, 0.2 mM of each dNTP (Amersham Pharmacia Biotech,Piscataway, N.J.), 0.5 μM of the primer pair, and 1 μl purifiedchromosomal template DNA. Cycling conditions involve an initial templatedenaturation step of 5 minutes at 94° C., follow by 35 cycles ofdenaturation at 94° C. for 30 seconds, annealing at 50° C. for 15seconds, and primer extension at 68° C. for 4 minutes. The PCR productsare subjected to electrophoresis in 1% (w/v) agarose gels in 1×TAEbuffer (40 mM Tris-acetate, 1 mM EDTA), staining with a 1 μg/ml ethidiumbromide solution and viewing over UV light.

The primers used for eighteen genes (out of twenty one) are indicated inTable 2. Of these eighteen genes, three of them (NAV-H23, NAV-H41 andNAV-H71) are present in 83% of the B. hyodysenteriae strains tested;three of them (NAV-H24, NAV-H30 and NAV-H73) are present in 87% of thestrains tested, seven of them (NAV-H22, NAV-H32, NAV-H33, NAV-H37,NAV-H43, NAV-H64 and NAV-H69) are present in 91% of the strains tested,and three of them (NAV-H40, NAV-H44, and NAV-H45) are present in 100% ofthe strains tested. The remaining three genes are present in less than80% of the B. hyodysenteriae strains tested. The poor distribution ofthese genes makes them less useful as a vaccine subunit. For thisreason, further analysis of these genes has been abandoned.

TABLE 2 Gene Primer name Primer Sequence (5′-3′) NAV-H22 H22-F4AAACGTTTATATTTTATTTTATC (SEQ ID NO: 67) H22-R1308 AAACTTCCAAGTGATACC(SEQ ID NO: 68) NAV-H23 H23-F4 AAATATAAACCTACAAGCAG (SEQ ID NO: 69)H23-R2366 AATATTTCAGTTAATCTAAAATC (SEQ ID NO: 70) NAV-H24 H24-F19ACTTTAATCTTTGTATTAATTTTG (SEQ ID NO: 71) H24-R729TTGTTTTAATTTGATAATATCAG (SEQ ID NO: 72) NAV-H30 H30-F4AAAAAAATTATTTTATTAATATTTATATT (SEQ ID NO: 73) H30-R969TTCTCTTATAATCTTTACAGTTG (SEQ ID NO: 74) NAV-H32 H32-F4CATATTTCTGGTGATTCTC (SEQ ID NO: 75) H32-R564 TTTTTTGATAAATAAGTTTTTTATTTG(SEQ ID NO: 76) NAV-H33 H33-F4 TTTAATACTCCTATATTATTAATTATTT(SEQ ID NO: 77) H33-R396 AAGGAGAATCACCAGAAA (SEQ ID NO: 78) NAV-H37H37-F4 AATGATATTATTAAAGTGATAAA (SEQ ID NO: 79) H37-R825AAAATCTAATATAACGGATT (SEQ ID NO: 80) NAV-H40 H40-F16AAATATGCTTCCATTATAGG (SEQ ID NO: 81) H40-R1815 ACTTTTAGGAAGAAGTTTAAC(SEQ ID NO: 82) NAV-H41 H41-F19 TATATTTTCATTATATATTTATTAG(SEQ ID NO: 83) H41-R1067 CTAGGCATAGATTTTCCA (SEQ ID NO: 84) NAV-H43H43-F46 TTTGCCATGTCGGAAATTGCAG (SEQ ID NO: 85) H43-R1236TATTCTAGCACCGTCCATATC (SEQ ID NO: 86) NAV-H44 H44-F43GTATGTTTATATGCTCAGGATAC (SEQ ID NO: 87) H44-R2931 AACAGCAGCACTATCTTGTAA(SEQ ID NO: 88) H44-F80 CAGCAGCAACAAATAATACTACTG (SEQ ID NO: 89)H44-R929 TGAATATAAACACCTTCTCTCAAAG (SEQ ID NO: 90) NAV-H45 H45-F52AAAATGTCATTGGTAACTACTGTAAG (SEQ ID NO: 91) H45-R1595CTTGATAATCTGCCTTTAAACATAC (SEQ ID NO: 92) NAV-H62 H62-F69ATGTGAGGAAAAAACAGAAAG (SEQ ID NO: 93) H62-R866 TCATTACCAGAAAACCATACTC(SEQ ID NO: 94) NAV-H64 H64-F69 AGGAAATAAAGCTCCTGCTGCTTCAGC(SEQ ID NO: 95) H64-R253 GCATAGCAGCAACTTCAGAAGGTCCA (SEQ ID NO: 96)NAV-H66 H66-F114 CTTATTAATTGGTATAGGAAAACC (SEQ ID NO: 97) H66-R200AATCTATGTTCTTGATTTATTAGCC (SEQ ID NO: 98) NAV-H69 H69-F546AGAAGCTACTTTTGGACCTTGGCCTGT (SEQ ID NO: 99) H69-R662ACACAGTCAACACCAAGAGC (SEQ ID NO: 100) NAV-H71 H71-F568AAACAGCAGACTAGCTGGTG (SEQ ID NO: 101) H71-R773TGACCATTACTTACACCGGATACCCCA (SEQ ID NO: 102) H71-F37TTAATGACTATATCGCTTTCATACACTTTC (SEQ ID NO: 103) H71-R1241TCAATTCTTCCAGACATAAAATCAGTAAG (SEQ ID NO: 104) NAV-H73 H73-F37TATATAGAGTGGGTATCAGAAG (SEQ ID NO: 105) H73-R254 TCATAATGGTATTTACAAGATG(SEQ ID NO: 106)

pTrcHis Plasmid Extraction

Escherichia coli JM 109 clones harboring the pTrcHis plasmid (InvitrogemCarlsbad, Calif.) are streaked out from glycerol stock storage ontoLuria-Bertani (LB) agar plates supplemented with 100 mg/l ampicillin andincubated at 37° C. for 16 hours. A single colony is used to inoculate10 ml of LB broth supplemented with 100 mg/l ampicillin, and the brothculture is incubated at 37° C. for 12 hours with shaking. The entireovernight culture is centrifuged at 5,000×g for 10 minutes, and theplasmid contained in the cells is extracted using the QIAprep® SpinMiniprep Kit (Qiagen, Doncaster VIC), The pelleted cells are resuspendedwith 250 μl cell resuspension buffer P1 and then are lysed with theaddition of 250 μl cell lysis buffer P2. The lysed cells are neutralizedwith 350 μl neutralization buffer N3, and the precipitated cell debrisis pelleted by centrifugation at 20,000×g for 10 minutes. Thesupernatant is transferred to a spin column and centrifuged at 10,000×gfor 1 minute. After discarding the flow-through, 500 μl wash buffer PEis applied to the column and centrifuged as before. The flow-through isdiscarded, and the column is dried by centrifugation at 20,000×g for 3minutes. The plasmid DNA is eluted from the column with 100 μl elutionbuffer EB. The purified plasmid is quantified using a Dynaquan™ DNAfluorometer (Hoefer, San Francisco, Calif.), and the DNA concentrationis adjusted to 100 μl by dilution with TE buffer. The purified pTrcHisplasmid is stored at −20° C.

Vector Preparation

Two μg of the purified pTrcHis plasmid is digested at 37° C. for 1-4hours in a total volume of 50 μl containing 5 U of two restrictionenzymes in 100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 1 mM DTTand 100 μg/ml BSA. The particular pair of restriction enzymes useddepends on the sequence of the primers and the sequence of the ORF; thegoal being to use primers that would not cut the ORFs. The restrictedvector is verified by electrophoresing 1 μl of the digestion reactionthrough a 1% (w/v) agarose gel in 1×TAE buffer at 90V for 1 hour. Theelectrophoresed DNA is stained with 1 μg/ml ethidium bromide and isviewed over ultraviolet (UV) light.

Linearised pTrcHis vector is purified using the UltraClean® PCR Clean-upKit (Mo Bio Laboratories, Carlsbad, Calif.), Briefly, the restrictionreaction (50 μl) is mixed with 250 μl SpinBind buffer B1, and the entirevolume is added to a spin-colunm. After centrifugation at 8,000×g for 1minute, the flow-through is discarded and 300 μl SpinClean buffer B2 isadded to the column, The column is centrifuged as before, and theflow-through is discarded before drying the column at 20,000×g for 3minutes. The purified vector is eluted from the column with 50 μl TEbuffer. Purified linear vector is quantified using a fluorometer, andthe DNA concentration is adjusted to 50 μl/ml by dilution with TEbuffer, The purified restricted vector is stored at −20° C.

Primer Design for Insert Preparation

Primer pairs are designed to amplify as much of the coding region of thetarget gene as possible using genomic DNA as the starting point. Allprimers sequences include terminal restriction enzyme sites to enablecohesive-end ligation of the resultant amplicon into the linearisedpTrcHis vector. The primers are tested using Amplify 1.2 (University ofWisconsin, Madison, Wis.) and the theoretical amplicon sequence isinserted into the appropriate position in the pTrcHis vector sequence.Deduced translation of the chimeric pTrcHis expression cassette isperformed using Vector NTI version 6 (InforMax) to confirm that the geneinserts would be in the correct reading frame. Table 3 also provides thegene size, the protein size, the predicted molecular weight of thenative protein in daltons and the predicted pI of the protein. It isnoted that the histidine-fusion of the recombinant protein addsapproximately 4 kDa to the native protein's predicted molecular weight.

TABLE 3 Gene size Protein size Predicted MW of Predicted Gene (bp) (aa)native protein (Da) pI NAV-H40 1815 605 97,733 9.4853 NAV-H41 1068 35639,870 5.2168 NAV-H44 2940 980 113,722 5.1864 NAV-H62 1014 338 376424.3944 NAV-H64 1011 337 36468 4.4953 NAV-H66 264 88 10629 9.3027 NAV-H691080 360 41525 5.7123 NAV-H73 258 86 10527 9.5920

Amplification of the Gene Inserts

Using genomic DNA, all target gene inserts are amplified by PCR in a 100μl total volume using Taq DNA polymerase (Biotech International) and PfuDNA polymerase (Promega, Madison, Wisc.). The amplification mixtureconsists of 1×PCR buffer (containing 1.5 mM of MgCl₂), 1 U of Tag DNApolymerase, 0.01 U Pfu DNA polymerase, 0.2 mM of each dNTP (AmershamPharmacia Biotech), 0.5 μM of the appropriate primer pair and 1 μl ofpurified chromosomal DNA. The chromosomal DNA is prepared from the sameB. hyodysenteriae strain used for genome sequencing. Cycling conditionsinvolve an initial template denaturation step of 5 minutes at 94° C.,followed by 35 cycles of denaturation at 94° C. for 30 seconds,annealing at 50° C. for 15 seconds, and primer extension at 68° C. for 4minutes, The PCR products are subjected to electrophoresis in 1% (w/vagarose gels in 1×TAE buffer, are stained with a 1 μl/ml ethidiumbromide solution and are viewed over UV light. After verifying thepresence of the correct size PCR product, the PCR reaction is purifiedusing the UltraClean® PCR Clean-up Kit (Mo Bio Laboratories, Carlsbad,Calif.), The PCR reaction (100 μl) is mixed with 500 μl SpinBind bufferB1, and the entire volume is added to a spin-column. Aftercentrifugation at 8,000×g for 1 minute, the flow-through is discarded,and 300 μl SpinClean buffer B2 is added to the column. The column iscentrifuged as before and the flow-through is discarded before dryingthe column at 20,000×g for 3 minutes. The purified vector is eluted fromthe column with 1000 μl TE buffer.

Restriction Enzyme Digestion of the Gene Inserts

Thirty μl of the purified PCR product are digested in a 50 μl totalvolume with 1 U of each restriction enzyme compatible with the terminalrestriction endonuclease recognition site determined by the cloningoligonucleotide primer. The restriction reaction consists of 100 mMTris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 1 mM DTT and 100 μg/ml BSAwith 1 U of each restriction enzyme at 37° C. for 1-4 hours, Thedigested insert DNA are purified using the UltraClean® PCR Clean-up Kit(see above), Purified digested insert DNA are quantified using thefluorometer, and the DNA concentration is adjusted to 20 μg/ml bydilution with TE buffer, The purified restricted insert DNA are usedimmediately for vector ligation.

Ligation of the Gene Inserts into the pTrcHis Vector

Ligation reactions are all performed in a total volume of 20 μl. Onehundred ng of linearised pTrcHis is incubated with 20 ng of restrictedinsert at 16° C. for 16 hours in 30 mM Tris-HCl (pH 7.8), 10 mM MgCl₂,10 mM DTT and 1 mM ATP containing 1 U of T4 DNA ligase (Promega). Anidentical ligation reaction containing no insert DNA is also included asa vector re-circularisation negative control. The appropriaterestriction enzyme is used for each reaction.

Transformation of pTrcHis Ligations into E. Coli Cells

Competent E. coli JM109 (Promega) cells are thawed from −80° C. storageon ice and then 50 μl of the cells are transferred into ice-cold 1.5 mlmicrofuge tubes containing 5 μl of the overnight ligation reactions(equivalent to 25 ng of pTrcHis vector). The tubes are mixed by gentlytapping the bottom of each tube on the bench and left on ice for 30minutes. The cells are then heat-shocked by placing the tubes into a 42°C. water bath for 45 seconds before returning the tube to ice for 2minutes. The transformed cells are recovered in 1 ml LB broth for 1 hourat 37° C. with gentle mixing. The recovered cells are harvested at2,500×g for 5 minutes, and the cells are resuspended in 50 μl of freshLB broth. The entire 50 μl of resuspended cells are spread evenly onto aLB agar plate containing 100 mg/l ampicillin using a sterile glass rod.Plates are incubated at 37° C. for 16 hours.

Detection of Recombinant pTrcHis Constructs in E. Coli by PCR

Twelve single transformant colonies for each construct are streaked ontofresh LB agar plates containing 100 mg/l ampicillin and incubated at 37°C. for 16 hours. A single colony from each transformation event isresuspended in 50 μl of TE buffer and is boiled for 1 minute. Two μl ofboiled cells are used as template for PCR. The amplification mixtureconsists of 1×PCR buffer (containing 1.5 mM of MgCl₂), 1 U of Taq DNApolymerase, 0.2 mM of each dNTP, 0.5 μM of the pTrcHis-F primer(5′-CAATTTATCAGACAATCTGTGTG-3′ SEQ ID NO: 107) and 0.5 μM of thepTrcHis-R primer (5′-TGCCTGGCAGTTCCCTACTCTCG-3′ SEQ ID NO: 108). Cyclingconditions involve an initial template denaturation step of 5 minutes at94° C., followed by 35 cycles of denaturation at 94° C. for 30 seconds,annealing at 60° C. for 15 seconds, and a primer extension at 72° C. for1 minute. The PCR products are subjected to electrophoresis in 1% (w/v)agarose gels in 1×TAE buffer, are stained with a 1 μg/ml ethidiumbromide solution and are viewed over UV light. Cloning of the variousinserts into the pTrcHis expression vector produces recombinantconstructs of various sizes.

Pilot Expression of Recombinant His-Tagged Proteins

Five to ten isolated colonies of recombinant pTrcHis construct in E.coli JM109 are inoculated into 3 ml LB broth in a 5 ml tube containing100 mg/l ampicillin and 1 mM IPTG and incubated at 37° C. for 16 hourswith shaking. The cells are harvested by centrifugation at 5,000×g for10 minutes at 4° C. The supernatant is discarded, and each pellet isresuspended with 10 μl Ni—NTA denaturing lysis buffer (100 mM NaH₂PO₄,10 mM Tris-HCl, 8 M urea, pH 8.0). After vortexing the tube for 1minute, the cellular debris is pelleted by centrifugation at 10,000×gfor 10 minute at 4° C. The supernatant is transferred to a new tube andstored at −20° C. until analysis.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-Page)

SDS-PAGE analysis of protein is performed using a discontinuousTris-glycine buffer system. Thirty μl of protein sample are mixed with10 μl 4× sample treatment buffer (250 mM Tris-HCl (pH 6.0), 8% (w/v)SDS, 200 mM OTT, 40% (v/v) glycerol and 0.04% (w/v) bromophenol blue).Samples are boiled for 5 minutes immediately prior to loading 10 μl ofthe sample into wells in the gel. The gel comprises a stacking gel (125mM Tris-HCl ph 6.8, 4% w/v acylamide, 0.15% w/v bis-acrylamide and 0.1%w/v SDS) and a separating gel (375 mM Tris-HCl pH 8.8, 12% w/vacylamide, 0.31% w/v bis-acrylamide and 0.1% SDS). These gels arepolymerised by the addition of 0.1% (v/v) TEMED and 0.05% (w/v) freshlyprepared ammonium sulphate solution and cast into the mini-Protean® dualslab cell (Bio-Rad, Hercules, Calif.). Samples are run at 150 V at roomtemperature (RT) until the bromophenol blue dye reaches the bottom ofthe gel. Pre-stained molecular weight standards are electrophoresed inparallel with the samples in order to allow molecular weightestimations. After eleetrophoresis, the gel is immediately stained usingCoomassie Brilliant Blue G250 (Bio-Rad) or is subjected toelectro-transfer onto nitrocellulose membrane for Western blotting.

Western Blot Analysis

Electrophoretic transfer of separated proteins from the SDS-PAGE gel tonitrocellulose membrane is performed using the Towbin transfer buffersystem. After electrophoresis, the gel is equilibrated in transferbuffer (25 mM Tris, 192 m/M glycine, 20% v/v methanol, pH 8.3) for 15minutes. The proteins in the gel are electro-transferred tonitrocellulose membrane (Protran, Schleicher and Schuell BioScience,Inc., Keene, N.H.) using the mini-Protean® transblot apparatus (Bio-Rad)at 30 V overnight at 4° C. The freshly transferred nitrocellulosemembrane containing the separated proteins is blocked with 10 ml ofTris-buffered saline (TBS) containing 5% (w/v) skim milk powder for 1hour at room temperature. The membrane is washed with TBS containing0.1% (v/v) Tween® 20 (TBST) and then is incubated with 10 mL mouseanti-his antibody (diluted 5,000-fold with TBST) for 1 hour at roomtemperature. After washing three times for 5 minutes with TBST, themembrane is incubated with 10 mL goat anti-mouse IgG (whole molecule)-APdiluted 5,000-fold in TBST for 1 hour at RT. The membrane is developedusing the Alkaline Phosphatase Substrate Kit (Bio-Rad). The developmentreaction is stopped by washing the membrane with distilled water. Themembrane is then dried and scanned for presentation.

Verification of Reading Frame of the Recombinant pTrcHis Constructs byDirect Sequence Analysis

Two transformant clones for each construct which produced the correctsized PCR products are inoculated into 10 ml LB broth containing 100mg/l ampicillin and incubated at 37° C. for 12 hours with shaking. Theentire overnight cultures are centrifuged at 5,000×g for 10 minutes, andthe plasmid contained in the cells are extracted using the QIAprep® SpinMiniprep Kit as described previously. The purified plasmid is quantifiedusing a fluorometer. Both purified plasmids are subjected to automateddirect sequencing of the pTrcHis expression cassette using the pTrcHis-Fand pTrcHis-R primers. Each sequencing reaction is performed in a 10J.11 volume consisting of 200 ng of plasmid DNA, 2 pmol of primer, and 4J.11 of the ABI PRISM™ BigDye® Terminator Cycle Sequencing ReadyReaction Mix (PE Applied Biosystems, Foster City, Calif.). Cyclingconditions involve a 2 minute denaturing step at 96° C., followed by 25cycles of denaturation at 96° C. for 10 seconds, and a combined primerannealing and extension step at 60° C. for 4 minutes. Residual dyeterminators are removed from the sequencing products by precipitationwith 95% (v/v) ethanol containing 85 mM sodium acetate (pH 5.2), 3 mMEDTA (pH 8), and vacuum dried. The plasmids are sequenced in duplicateusing each primer. Sequencing products are analysed using an ABI 373ADNA Sequencer (PB Applied Biosystems). Nucleotide sequencing of thepTrcHis is performed to verify that the expression cassette is in thecorrect reading frame for each constructs.

Expression and Purification of Recombinant His-Tagged Proteins

A single colony of the recombinant pTrcHis construct in E. coli JM 109is inoculated into 50 ml LB broth in a 250 ml conical flask containing100 mg/l ampicillin and incubated at 37° C. for 16 hours with shaking. A2 l conical flask containing 1 l of LB broth supplemented with 100 mg/lampicillin is inoculated with 10 ml of the overnight culture andincubated at 37° C. until the optical density of the cells at 600 nm is0.5 (approximately 3-4 hours). The culture is then induced by addingIPTG to a final concentration of 1 mM, and the cells are returned to 37°C. with shaking. After 5 hours of induction, the culture is transferredto 250 ml centrifuge bottles, and the bottles are centrifuged at 5,000×gfor 20 minutes at 4° C. The supernatant is discarded, and each pellet isresuspended with 8 ml Ni—NTA denaturing lysis buffer (100 mM NaH₂PO₄, 10mM Tris-HCl, 8 M urea, pH 8.0). The resuspended cells are stored at −20°C. overnight.

The cell suspension is removed from −20° C. storage and thawed on ice.The cell lysate is then sonicated on ice 3 times for 30 seconds with 1minute incubation on ice between sonication rounds. The lysed cells arecleared by centrifugation at 20,000×g for 10 minutes at 4° C., and thesupernatant is transferred to a 15 ml column containing a 0.5 ml bedvolume of Ni—NTA agarose resin (Qiagen). The recombinant His₆-taggedprotein is allowed to bind to the resin for 1 hour at 4° C. withend-over-end mixing. The resin is then washed with 30 ml of Ni—NTAdenaturing wash buffer (100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH6.3) before elution with 12 ml of Ni—NTA denaturing elution buffer (100mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 4.5). Four 3 ml fractions ofthe eluate are collected and stored at 4° C. Thirty μl of each eluate istreated with 10 μl of 4× sample treatment buffer and boiled for 5minutes. The samples are subjected to SDS-PAGE and stained withCoomassie Brilliant Blue G250 (Bio-Rad). The stained gel is equilibratedin distilled water for 1 hour and dried between two sheets of celluloseovernight at RT.

Expression of the selected recombinant E. coli clones is performed inmedium-scale to generate sufficient recombinant protein for vaccinationof mice (see below).

Dialysis and Lyophilisation of the Purified Recombinant His-TaggedProtein

The eluted proteins are pooled and transferred into a hydrated dialysistube (Spectrum Laboratories, Inc., Los Angeles, Calif.) with a molecularweight cut-off (MWCO) of 3,500 Da. A 200 μl aliquot of the pooled eluateis taken and quantified using a commercial Protein Assay (Bio-Rad). Theproteins are dialysed against 21 of distilled water at 4° C. withstirring. The dialysis buffer is changed 8 times at 12-hourly intervals.The dialysed proteins are transferred from the dialysis tube into a 50ml centrifuge tubes (40 ml maximum volume), and the tubes are placed at−80° C. overnight. Tubes are placed into a MAXI freeze-drier(Heto-Holten, Allerod, Denmark) and lyophilised to dryness. Thelyophilised proteins are then re-hydrated with PBS to a calculatedconcentration of 2 mg/ml and stored at −20° C. Following dialysis andlyophilisation, stable recombinant antigen is successfully produced.

Eight of the eighteen genes are successfully cloned into the E. coliExpression System and recombinant protein can be expressed stabily fromthese clones.

Serology Using Purified Recombinant Protein

Twenty μg of purified recombinant protein is loaded into a 7 cm IEFwell, electrophoresed through a 10% (w/v) SDS-PAGE gel, andelectro-transferred to nitrocellulose membrane. The membrane is blockedwith TBS-skim milk (5% w/v) and assembled into the multi-screenapparatus (Bio-Rad). The wells are incubated with 100 μl of diluted pigserum (100-fold) for 1 hour at room temperature. The pig serum isobtained from high health status pigs (n=3), experimentally challengedpigs showing clinical SD (n=5), naturally infected seroconverting pigs(n=5), and pigs recovered from natural infection (n=4). The membranethen is removed from the apparatus and washed three times with TBST(0.1% v/v) before incubating with 10 ml of goat anti-swine IgG (wholemolecule)-AP (5,000-fold) for 1 hour at RT. The membrane is washed threetimes with TBST before color development using an Alkaline PhosphataseSubstrate Kit (Bio-Rad). The membrane is washed with tap water whensufficient development has occurred, dried and scanned for presentation.

The reactivity of the pig serum obtained from animal of differing healthstatus is shown in the table below. All proteins are recognised by 100%of the panel of serum thus indicating that the genes are expressed invivo and that they are able to induce a systemic immune responsefollowing exposure to the spirochaete.

TABLE 4 Gene distribution and serologic reactivity of the eightsuccessfully expressed B. hyodysenteriae vaccine candidates. GeneDistribution (%) Serology (%) NAV-H40 100 100% NAV-H41 83 100% NAV-H44100 100% NAV-H62 96 100% NAV-H64 91 100% NAV-H66 96 100% NAV-H69 91 100%NAV-H73 87 100% The gene distribution was analysed by PCR using a panelof 23 different strains. Serology was performed using 19 serum samplesfrom five different categories of disease.

Vaccination of Mice Using the Purified Recombinant his-Tagged Proteins

For each of the purified recombinant his-tagged proteins, ten mice aresystemically and orally immunized to determine whether the recombinantprotein would be immunogenic. The recombinant protein is emulsified with30% (v/v) water in oil adjuvant and injected intramuscularly into thequadraceps muscle of ten mice (Balb/cJ: 5 weeks old males). All micereceive 100 μg of protein in a total volume of 100 μl. Three weeks afterthe first vaccination, all mice receive a second intramuscularvaccination identical to the first vaccination. All mice are killed twoweeks after the second vaccination. Sera are obtained from the heart atpost-mortem and tested in Western blot analysis for antibodies againstcellular extracts of B. hyodysenteriae.

Western Blot Analysis

Twenty μg of purified recombinant protein is loaded into a 7 cm IEFwell, electrophoresed through a 10% (w/v) SDS-PAGE gel, andelectro-transferred to nitrocellulose membrane. The membrane is blockedwith TBS-skim milk (5% w/v) and assembled into the multi-screenapparatus (Bio-Rad). The wells are incubated with 100 μl of dilutedmouse serum (100-fold) for 1 hour at room temperature. The membrane isremoved from the apparatus and washed three times with TBST (0.1% v/v)before incubating with 10 ml of goat anti-mouse IgG (whole molecule)-AP(5,000-fold) for 1 hour at room temperature. The membrane is washedthree times with TBST before color development using an AlkalinePhosphatase Substrate Kit (Bio-Rad). The membrane is washed with tapwater when sufficient development has occurred, dried and scanned forpresentation.

Western blot analysis shows a significant increase in antibodyreactivity in the mice towards the recombinant vaccine antigensfollowing vaccination. All the mice recognise recombinant proteins whichare similar in molecular weight to that of the coomassie blue stainedpurified recombinant proteins. These experiments provide evidence thatthe recombinant proteins are immunogenic when used to vaccinate mice andthat the vaccination protocol employed can induce specific circulatingantibody titres against the antigen. The results indicate that therecombinant proteins can be useful in an effective vaccine for animalspecies from being colonised by B. hyodysenteriae.

Vaccination of Pigs Using the Purified Recombinant his-Tagged Proteins

For each of the purified recombinant his-tagged proteins, tensero-negative pigs are injected intramuscularly with 1 mg of theparticular antigen in 1 ml vaccine volume. The antigen is emulsifiedwith an equal volume of a water-in-oil adjuvant. The pigs are vaccinatedat three weeks of age and again at six weeks of age. A second group often sero-negative pigs is used as negative controls and are leftunvaccinated. All pigs are challenged with 100 ml of an active B.hyodysenteriae culture (˜10⁹ cells/ml) at eight weeks of age, and thepigs are observed for clinical signed of swine dysentery during theexperiment (up to six weeks post-challenge) and at post-mortenexamination.

Diagnostic Kit

Serum is obtained from pigs in a piggery with known infection of B.hyodysenteriae, from pigs known to have not been infected with B.hyodysenteriae, and from pigs in piggery with unknown infection with B.hyodysenteriae. Twenty μg of purified recombinant protein is loaded intoa 7 cm IEF well, electrophoresed through a 10% (w/v) SDS-PAGE gel, andelectro-transferred to nitrocellulose membrane. The membrane is blockedwith TBS-skim milk (5% w/v) and assembled into the multi-screenapparatus (Bio-Rad). The wells are incubated with 100 μl of diluted pigserum (100-fold) for 1 hour at room temperature. The membrane then isremoved from the apparatus and washed three times with TBST (0.1% v/v)before incubating with 10 ml of goat anti-swine IgG (whole molecule)-AP(5,000-fold) for 1 hour at room temperature. The membrane is washedthree times with TBST before color development using an AlkalinePhosphatase Substrate Kit (Bio-Rad). The membrane is washed with tapwater when sufficient development has occurred, dried and scanned forpresentation. One can determine if pigs are infected with B.hyodysenteriae by comparing the results to the positive and negativecontrol.

While this invention has been described with a reference to specificembodiments, it will be obvious to those of ordinary skill in the artthat variations in these methods and compositions may be used and thatit is intended that the invention may be practiced otherwise than asspecifically described herein. Accordingly this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the claims.

The invention claimed is:
 1. An isolated polypeptide comprising the fulllength of the amino acid sequence SEQ ID NO: 60 and a heterologouspolypeptide that permits the detection, isolation, solubilization, orstabilization of the isolated polypeptide.
 2. An isolated polypeptidecomprising a sequence that is at least 70% homologous to a polypeptidewith the amino acid sequence SEQ ID NO: 60, and heterologous polypeptidethat permits the detection, isolation, solubilization, or stabilizationof the isolated polypeptide.
 3. An isolated polypeptide comprising asequence that is at least 80% homologous to a polypeptide with the aminoacid sequence SEQ ID NO: 60, and a heterologous polypeptide that permitsthe detection, isolation, solubilization, or stabilization of theisolated polypeptide.
 4. An isolated polypeptide comprising a sequencethat is at least 90% homologous to a polypeptide with the amino acidsequence SEQ ID NO: 60, and a heterologous polypeptide that permits thedetection, isolation, solubilization, or stabilization of the isolatedpolypeptide.
 5. An immunogenic composition comprising the polypeptideclaim
 1. 6. A method of generating an immune response to a Brachyspirainfection in an animal comprising administering to said animal thepolypeptide of claim
 1. 7. An immunogenic composition comprising thepolypeptide of claim
 2. 8. A method of generating an immune response toa Brachyspira infection in an animal comprising administering to saidanimal the polypeptide of claim
 2. 9. An immunogenic compositioncomprising the polypeptide of claim
 3. 10. A method of generating animmune response to a Brachyspira infection in an animal comprisingadministering to said animal the polypeptide of claim
 3. 11. Animmunogenic composition comprising the polypeptide of claim
 4. 12. Amethod of generating an immune response to a Brachyspira infection in ananimal comprising administering to said animal the polypeptide of claim4.